http://2008.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Aronlau2008.igem.org - User contributions [en]2024-03-29T12:52:30ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T04:04:07Z<p>Aronlau: /* Strains */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview6.jpg|500 px]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/File:Genomic_Gateway_Overview6.jpgFile:Genomic Gateway Overview6.jpg2008-10-30T04:03:43Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T04:03:37Z<p>Aronlau: /* Strains */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview6.jpg|900 px]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/File:Genomic_Gateway_Overview5.jpgFile:Genomic Gateway Overview5.jpg2008-10-30T04:02:44Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T04:00:16Z<p>Aronlau: /* Strains */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview5.jpg|900 px]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/File:Genomic_Gateway_Overview4.jpgFile:Genomic Gateway Overview4.jpg2008-10-30T03:59:57Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T03:59:49Z<p>Aronlau: /* Strains */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview4.jpg|900 px]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/File:Genomic_Gateway_Overview3.jpgFile:Genomic Gateway Overview3.jpg2008-10-30T03:59:34Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T03:59:27Z<p>Aronlau: /* Strains */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview3.jpg|900 px]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T03:58:43Z<p>Aronlau: /* Strains */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview2.jpg|900 px]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T03:58:19Z<p>Aronlau: /* Strains */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview2.jpg|600 px]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/File:Genomic_Gateway_Overview2.jpgFile:Genomic Gateway Overview2.jpg2008-10-30T03:57:59Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T03:57:41Z<p>Aronlau: /* Strains */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview2.jpg]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/File:Genomic_Gateway_Overview.jpgFile:Genomic Gateway Overview.jpg2008-10-30T03:56:57Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/File:.ucbphagemid.pngFile:.ucbphagemid.png2008-10-30T03:39:54Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPhagemidTeam:UC Berkeley/GatewayPhagemid2008-10-30T03:39:47Z<p>Aronlau: /* picture */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Phagemid Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The phagemid scheme was motivated by a desire to further streamline the protocol for performing Gateway ''in vivo''. Like the genomic scheme, this method utilizes positive selection to isolate the desired product. However, it improves on this scheme because the lysis and infection steps replace mini-preps and transformations without requiring additional devices.<br />
<br />
This scheme utilizes two methods of positive selection: conditional origins of replication (''oriV'' and ''oriR6K'') and the ability to be packaged into and transported by a phage. When induced with arabinose, the phagemid (plasmid that can be packaged and transported by a phage) will be copied and packaged in phages that will subsequently lyse the cell. The resulting lysate containing phages holding the plasmid of interest can be used to transport the plasmid into other cells via infection.<br />
<br />
==Details about Plasmids and Strains==<br />
===Plasmids===<br />
The critical plasmid necessary for this scheme is the phagemid, which contains the assembly vector components as well as the device allowing the plasmid to be packaged and transported by a phage. The <br />
<br />
===picture===<br />
[[Image:.ucbphagemid.png]]<br />
[[Image:UCB_phagemidoverview.png]]<br />
<br />
==General Procedure==<br />
[[Image:ucbphagemidoverview2.png]]<br />
<br />
==Conclusion==<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/LysisDevice" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T03:36:11Z<p>Aronlau: /* Plasmids */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|250 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview.jpg]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayGenomicTeam:UC Berkeley/GatewayGenomic2008-10-30T03:35:56Z<p>Aronlau: /* Plasmids */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Genomic Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The ''in vivo'' plasmid based gateway scheme was successful and produced the desired product, but it also yielded a considerable amount of background. Closer analysis of the side products revealed that the background resulted from ''ccdB'' mutations that arose when the gene replicated and recombined ''in vivo''. In an effort to circumvent background resulting from mutations in a negative selection gene, we decided to use positive selection. This way, we would be able to select for the desired products rather than selecting against unwanted side products and unreacted starting materials.<br />
<br />
In order to implement positive selection, we used conditional origins of replication on our plasmids. These origins of replication will only be functional and allow plasmid replication if a specific protein is expressed in the cell. In our design, the assembly vector with an ''oriR6K'' origin (requires the protein Pi) is integrated into the genome. The gene of interest alongside an ''oriV'' origin (requires the protein TrfA) can then recombine with the genome. In this manner, the gene of interest can be moved to an assembly vector by inserting the entry vector into the genome. We could allow selective replication of the plasmids in our scheme by performing the reaction in a cell expressing TrfA and then transforming the resulting mixture of plasmids into a cell expressing ‘’pir’’.<br />
<br />
We also incorporated our lysis device into our new entry vector in order to simplify the experimental protocol for this scheme by eliminating mini-preps.<br />
<br />
==Details about Plasmids and Strains==<br />
<br />
===Plasmids===<br />
The entry plasmid in this scheme contains both the part of interest and an ''oriV'' origin of replication within the attL recombination sites, thereby allowing this origin to be transferred to the desired product. Since the entry plasmid has no constitutive origin of replication, it can only replicate in the presence of the protein TrfA. With the exception of the change in type and placement of the origin of replication, the entry plasmid maintains the same features as the traditional entry plasmid, pBca1256.<br />
<br />
[[Image:OriV entry vector.jpg|150 px]]<br />
<br />
In variations on this scheme where Xis and Int are not integrated into the genome, they are placed prior to the'' attL1'' site under the control of a temperature sensitive promoter. In an effort to streamline this method, we also considered a variation where the lysis device was placed before Xis and Int in the entry plasmid.<br />
<br />
===Strains===<br />
This scheme utilizes strains that include the assembly vector in the genome. The traditional double-antibiotic assembly vector has been modified to include an ''oriR6K'' origin of replication (which can be induced by the Pi protein) in place of a constitutively active replication origin. Since the integrated vector includes the ''attR2'' sites that usually flank the vector region of the assembly plasmid, the ''attL1'' sites in the entry plasmid can recombine with the genome to produce the desired product. <br />
<br />
In addition to the integrated assembly vector, the strains also include TrfA in the genome to allow the replication of the entry vector containing an ''oriV'' origin. Additionally, one variation on this scheme has ''xis'' and ''int'' placed in the the genome to catalyze the recombination reaction.<br />
<br />
[[Image:Genomic Gateway Overview.jpg]]<br />
<br />
==General Procedure==<br />
In this scheme, the ''oriV'' version of the entry plasmid can simply be transformed into the integrated strain containing the desired assembly vector and TrfA. In order to catalyze the reaction, Xis and Int must be present either on the plasmid or in the genome. <br />
<br />
Once the entry plasmid has been transformed and the reaction has been catalyzed, the desired product will be able to replicate in the cytoplasm of the cell using the ''oriV'' origin that was transferred with the part from the entry vector to the assembly vector. The product can be released from the cell by inducing our self-lysis device (which can be installed in the entry vector) or by doing a mini-prep.<br />
<br />
Once the plasmids in the cell's cytoplasm have been obtained, they need to be transformed into cells that express the Pi protein. This will allow for the selective replication of the desired product, which contains an ''oriR6K'' origin obtained from the assembly vector that was originally in the genome. <br />
<br />
The potential source of background in this scheme is the unreacted entry plasmid. However, this plasmid does not have an ''oriR6K'' origin (it only contains an ''oriV'' origin), so it is unable to replicate in cells that express Pi instead of TrfA. Differential antibiotic markers would assist in screening against the bleed-through of the starting plasmid, but only positive selection can eliminate the need to screen for contransformants (which contain both the starting plasmid in addition to the desired product).<br />
<br />
==Conclusion==<br />
With the incorporation of the lysis device, the genomic Gateway scheme promises to be an improvement to the plasmid based Gateway scheme because it eliminates unwanted background by using positive selection. However, the protocol for this scheme still requires that the plasmid mixture is transformed into another cell strain to select for the desired product. We sought to further streamline this scheme by utilizing phagemids, which can lyse the cell and infect other cells without the need for either mini-preps or transformation.<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/File:OriV_entry_vector.jpgFile:OriV entry vector.jpg2008-10-30T03:33:51Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/File:Ucbphagemidoverview2.pngFile:Ucbphagemidoverview2.png2008-10-30T03:31:27Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPhagemidTeam:UC Berkeley/GatewayPhagemid2008-10-30T03:31:18Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Phagemid Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The phagemid scheme was motivated by a desire to further streamline the protocol for performing Gateway ''in vivo''. Like the genomic scheme, this method utilizes positive selection to isolate the desired product. However, it improves on this scheme because the lysis and infection steps replace mini-preps and transformations without requiring additional devices.<br />
<br />
This scheme utilizes two methods of positive selection: conditional origins of replication (''oriV'' and ''oriR6K'') and the ability to be packaged into and transported by a phage. When induced with arabinose, the phagemid (plasmid that can be packaged and transported by a phage) will be copied and packaged in phages that will subsequently lyse the cell. The resulting lysate containing phages holding the plasmid of interest can be used to transport the plasmid into other cells via infection.<br />
<br />
==Details about Plasmids and Strains==<br />
==General Procedure==<br />
[[Image:ucbphagemidoverview2.png]]<br />
<br />
==Conclusion==<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/LysisDevice" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/File:Ucbphagemidoverview1.pngFile:Ucbphagemidoverview1.png2008-10-30T03:29:30Z<p>Aronlau: </p>
<hr />
<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPhagemidTeam:UC Berkeley/GatewayPhagemid2008-10-30T03:29:21Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Phagemid Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The phagemid scheme was motivated by a desire to further streamline the protocol for performing Gateway ''in vivo''. Like the genomic scheme, this method utilizes positive selection to isolate the desired product. However, it improves on this scheme because the lysis and infection steps replace mini-preps and transformations without requiring additional devices.<br />
<br />
This scheme utilizes two methods of positive selection: conditional origins of replication (''oriV'' and ''oriR6K'') and the ability to be packaged into and transported by a phage. When induced with arabinose, the phagemid (plasmid that can be packaged and transported by a phage) will be copied and packaged in phages that will subsequently lyse the cell. The resulting lysate containing phages holding the plasmid of interest can be used to transport the plasmid into other cells via infection.<br />
<br />
==Details about Plasmids and Strains==<br />
==General Procedure==<br />
[[Image:ucbphagemidoverview1.png]]<br />
<br />
==Conclusion==<br />
<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/LysisDevice" class="titleIcon"><br />
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</html></div>Aronlauhttp://2008.igem.org/File:Ucbphagemidoverview.pngFile:Ucbphagemidoverview.png2008-10-30T03:27:26Z<p>Aronlau: </p>
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<div></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPhagemidTeam:UC Berkeley/GatewayPhagemid2008-10-30T03:27:18Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Phagemid Based Gateway'''</font></div><br><br />
<br />
==Introduction==<br />
The phagemid scheme was motivated by a desire to further streamline the protocol for performing Gateway ''in vivo''. Like the genomic scheme, this method utilizes positive selection to isolate the desired product. However, it improves on this scheme because the lysis and infection steps replace mini-preps and transformations without requiring additional devices.<br />
<br />
This scheme utilizes two methods of positive selection: conditional origins of replication (''oriV'' and ''oriR6K'') and the ability to be packaged into and transported by a phage. When induced with arabinose, the phagemid (plasmid that can be packaged and transported by a phage) will be copied and packaged in phages that will subsequently lyse the cell. The resulting lysate containing phages holding the plasmid of interest can be used to transport the plasmid into other cells via infection.<br />
<br />
==Details about Plasmids and Strains==<br />
==General Procedure==<br />
[[Image:ucbphagemidoverview.png]]<br />
<br />
==Conclusion==<br />
<br />
<br />
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<a href="https://2008.igem.org/Team:UC_Berkeley/LysisDevice" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/ModelingTeam:UC Berkeley/Modeling2008-10-30T03:09:39Z<p>Aronlau: /* Governing Equations */</p>
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<div>__NOTOC__<br />
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<div style="text-align: center;"><font size="6">'''Modeling'''</font></div><br><br />
<br />
=='''Motivation'''==<br />
<br />
Since several phage systems and many promoters are currently in use. Understanding the important parameters of the system allows one to choose appropriate promoters and phage proteins to optimize lysis behavior. Therefore we have created a steady-state kinetics model to describe the system. Our model is in agreement with our experimental results and published data regarding the T4 and λ phage systems.<br />
<br />
=='''Introduction'''==<br />
<br />
The holin-lysozyme phage lysis device consists of three proteins: <br />
<br />
Holin - inner membrane-bound proteins that when complexed with other holin proteins, create holes in the membrane that allow lysozyme access to the periplasm. <br />
<br />
Antiholin - inner membrane-bound (λ) or cytoplasmic (T4) protein that binds to and inactivates holin, producing an inactive holin-antiholin dimer. In the λ phage system, this dimer becomes active after holin forms enough pores in the plasma membrane to cause a loss of proton motive force between the periplasm and the cytosol.<br />
<br />
Lysozyme - once holin forms pores in the inner membrane, lysozyme enters the periplasm and degrades peptidoglycan, resulting in cell lysis.<br />
<br />
[[Image:holinantiholin1.jpg]]<br />
<br />
=='''Governing Equations'''==<br />
Our lysis device consists of holin and lysozyme under an inducible promoter and antiholin under a constitutive promoter. The kinetics of our phage lysis device was modeled using first and second-order rate equations for the mRNAs and proteins of interest. The below equations describe our system <br />
<br />
[[Image:cdb1.jpg]]<br />
<br />
where P<sub>H</sub> represent the holin mRNA promoter strength as a function of arabinose concentration and P<sub>AH</sub> represents the antiholin mRNA promoter strength. γ<sub>mRNA,H</sub> and γ<sub>mRNA,AH</sub> represent the degradation rates for holin and antiholin mRNAs respectively and γ represents the protein degradation rate. k<sub>H</sub> and k<sub>AH</sub> represent the rate constants for holin and antiholin protein translation and k<sub>c</sub> and k<sub>u</sub> represent the coupling and uncoupling rates for the holin-antiholin dimer.<br />
<br />
=='''Transfer Function and Dimensionless Parameters'''== <br />
<br />
At steady state, <br />
<br />
[[Image:cdb2.jpg]] <br />
<br />
By making this assumption, the system of equations can be simplified into the following transfer function<br />
<br />
[[Image:cdb3.jpg]] <br />
<br />
Where the system can be divided into three dimensionless parameters which describe the behavior of the holin-antiholin dimer and the holin and antiholin proteins.<br />
<br />
[[Image:cdb4.jpg]] <br />
<br />
These dimensionless numbers can assist in the optimization of lysis device design because they describe the important parameters in the system.<br />
<br />
For example, Ω<sub>H</sub> consists of the rate constants for the formation of holin mRNA and protein divided by their degradation rates. Strong promoters on holin will increase the value of Ω<sub>H</sub>, while high protein degradation rates will decrease Ω<sub>H</sub>.<br />
<br />
Ω<sub>AH</sub> describes the rate constants and degradation rates for antiholin.<br />
<br />
Φ describes the importance of the coupling and uncoupling rates of holin-antiholin dimer as well as the degradation rate of the dimer.<br />
<br />
=='''Graphs'''==<br />
<br />
Physiologically relevant values for Ω<sub>H</sub>, Ω<sub>AH</sub> and Φ were estimated based on rate constants for similar proteins. This system was input into MatLab to produce the following graph. Since there is a degree of uncertainty in these estimates, the graph spans several orders of magnitude above and below our estimated values. For the MatLab code used to produce these graphs, please click here: [[Matlab code]]. <br />
<br />
[[Image:cdb5.jpg]]<br />
<br />
The literature indicates that at the time of lysis, cells infected with λ phage have approximately 1000 holin proteins<sup>1</sup>. Therefore, the critical concentration of holin (H<sub>c</sub>) was set at 1000 holin proteins per cell. <br />
<br />
The horizontal line at y=1 represents the critical holin concentration needed induce lysis. If the system produces more than the critical concentration of holin (>1000 free holin proteins), lysis is expected to occur.<br />
<br />
As one would expect by looking at the dimensionless value for holin (omega<sub>H</sub>), as the strength of the holin promoter increases, the amount of holin at steady state increases. <br />
<br />
The graph also shows that the system is not very sensitive to small amounts of antiholin. Larger amounts of antiholin push the system equilibrium to the left and would require a stronger promoter on holin or a weaker binding interaction between holin and antiholin to reach critical concentration. This is supported by the observation that in a system with no antiholin, lysis can occur with holin under a weaker promoter.<br />
<br />
Varying Φ, the dimensionless parameter that describes the coupling and uncoupling behavior of the holin-antiholin dimer, reveals that when the binding of holin-antiholin is stronger, a stronger promoter is required to reach the critical holin concentration.<br />
<br />
=='''Conclusions'''==<br />
There are several aspects of the model that agree with our experimental results and published data regarding phage lysis: <br />
<br />
1. Our experimental results show that at higher concentrations of arabinose, lysis occurs more readily. In our model, higher concentrations of arabinose increase the value of P<sub>H</sub> and thus increase the value of omega<sub>H</sub>. Our model predicts that as omega<sub>H</sub> increases, lysis is more likely to occur.<br />
<br />
2. Our experimental results showed that the T4 lysis device results in lysis at a lower concentration of arabinose than our λ lysis device when the systems are under the same promoter. λ antiholin and holin should have similar degradation rates because both are membrane-bound and only differ by two amino acids<sup>2</sup>. In contrast, T4 antiholin is an unstable cytoplasmic protein with a rapid degradation rate (t<sub>1/2</sub> = 2 min)<sup>3</sup>. Therefore γ should be larger in the T4 system, resulting in a larger value for Φ. This will shift the graph up and allow the system to reach the critical holin concentration more readily (see graph with Φ =400). <br />
<br />
3. Our model shows that systems with low values of omega<sub>AH</sub> (weaker antiholin mRNA or protein promoter) will lyse more readily than systems with large values of omega<sub>AH</sub>. This is in agreement with previously published results that show that in cells with little or no antiholin, lysis occurs more readily <sup>2</sup>. <br />
<br />
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<small>1. Christos G. Savva, Jill S. Dewey, John Deaton, Rebecca L. White, Douglas K. Struck, Andreas Holzenburg and Ry Young. The holin of bacteriophage lambda forms rings with large diameter. Molecular Microbiology 69(4), 784–793. 2008.<br />
<br />
2. Martin Steiner and Udo Bläs. Charged amino-terminal amino acids affect the lethal capacity of Lambda lysis proteins S107 and S105. Molecular Microbiology. 8(3), 525 - 533. Oct. 2006.<br />
<br />
3. Tram Anh T. Tran, Douglas K. Struck, and Ry Young*. The T4 RI Antiholin Has an N-Terminal Signal Anchor Release Domain That Targets It for Degradation by DegP. Journal of Bacteriology. 7618–7625. Nov. 2007. </small></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:07:17Z<p>Aronlau: /* Gateway Device in Entry Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named [https://static.igem.org/mediawiki/2008/b/b3/PK112245.png pK112245] and [https://static.igem.org/mediawiki/2008/f/f3/PK112246.png pK112246] respectively. A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was [https://static.igem.org/mediawiki/2008/8/85/PBca_1256.png pBca1256].<br><br />
<br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/7/76/PK112128.png pK112128]. <br> <br />
<br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named [https://static.igem.org/mediawiki/2008/2/2d/PK112247.png pK112247] and [https://static.igem.org/mediawiki/2008/a/ae/PK112248.png pK112248] respectively.<br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
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</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:06:28Z<p>Aronlau: /* Gateway Device in Assembly Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named [https://static.igem.org/mediawiki/2008/b/b3/PK112245.png pK112245] and [https://static.igem.org/mediawiki/2008/f/f3/PK112246.png pK112246] respectively. A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was [https://static.igem.org/mediawiki/2008/8/85/PBca_1256.png pBca1256].<br><br />
<br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/7/76/PK112128.png pK112128]. <br> <br />
<br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named [https://static.igem.org/mediawiki/2008/2/2d/PK112247.png pK112247] and [https://static.igem.org/mediawiki/2008/a/ae/PK112248.png pK112248] respectively (see links below).<br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:05:43Z<p>Aronlau: /* Gateway Device in Assembly Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named [https://static.igem.org/mediawiki/2008/b/b3/PK112245.png pK112245] and [https://static.igem.org/mediawiki/2008/f/f3/PK112246.png pK112246] respectively. A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/7/76/PK112128.png pK112128]. <br> <br />
<br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named [https://static.igem.org/mediawiki/2008/2/2d/PK112247.png pK112247] and [https://static.igem.org/mediawiki/2008/a/ae/PK112248.png pK112248] respectively (see links below).<br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:04:27Z<p>Aronlau: /* Gateway Device in Entry Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/7/76/PK112128.png pK112128]. <br> <br />
<br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named [https://static.igem.org/mediawiki/2008/2/2d/PK112247.png pK112247] and [https://static.igem.org/mediawiki/2008/a/ae/PK112248.png pK112248] respectively (see links below).<br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:04:03Z<p>Aronlau: /* Gateway Device in Entry Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/7/76/PK112128.png pK112128]. <br> <br />
<br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named [https://static.igem.org/mediawiki/2008/2/2d/PK112247.png pK112247] and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:03:14Z<p>Aronlau: /* Gateway Device in Entry Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/7/76/PK112128.png pK112128]. <br> <br />
<br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:01:15Z<p>Aronlau: /* Gateway Device in Entry Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/7/76/PK112128.png pK112128] (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:00:47Z<p>Aronlau: /* Gateway Device in Entry Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T03:00:22Z<p>Aronlau: /* Gateway Device in Entry Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/2/2d/PK112247.png|pK112128] (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:59:38Z<p>Aronlau: /* Gateway Device in Entry Vector: Plasmid Details */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is [https://static.igem.org/mediawiki/2008/2/2d/PK112247.png| pK112128] (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
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</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayOverviewTeam:UC Berkeley/GatewayOverview2008-10-30T02:56:10Z<p>Aronlau: /* The Natural Lambda Phage Recombination system */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Gateway Overview'''</font></div><br><br />
==Why Use Gateway?==<br />
<br />
The first step of the layered assembly scheme involves the transfer of biobrick parts from an entry vector to a double antibiotic assembly vector. Traditionally, this would require a fairly work-intensive protocol requiring digestion, gel purification, ligation, transformation, and plasmid isolation. In addition to being more time-consuming, the aforementioned procedure is also suboptimal because it is difficult to scale-up.<br />
<br />
The Gateway Cloning approach developed by [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html Invitrogen] offers a more efficient and convenient alternative for parts transfer. Their procedure involves the enzyme-catalyzed exchange of parts flanked by specific recombination sites. Experimentally, it is a one-pot, room-temperature reaction where the plasmids, buffer, water and enzymes are added together. After the addition of another enzyme and a short incubation period to terminate the reaction, the entire mixture can be transformed directly. This one-pot approach with a fewer steps is much more suitable for large-scale experimentation.<br />
<br />
Gateway is commonly used to facilitate the transfer of a single gene of interest from an entry clone to multiple destination vectors, as shown below. The efficiency and robustness of the Gateway mechanism are ideal for this application because once the gene of interest is cloned and confirmed in the entry vector, subsequent transfers using Gateway need not be confirmed again. Thus, it is ideal for use in the first step of layered assembly where a part may be transferred to one or more of the double antibiotic vectors required for the subsequent assembly steps assembly.<br />
<br />
[[Image:gatewayoverview.png|800px|thumb|center|A entry vector generated from any of the methods can be transferred to various different vectors using the Gateway method. <br>]]<br />
<br />
==Gateway Chemistry==<br />
<br />
===The Natural Lambda Phage Recombination system===<br />
<br />
In general, Gateway reactions in the lab involve the attB, attP, attL, and attR recombination sites and the integrase (Int), excisionase (Xis), and integration host factor (IHF) enzymes. These enzymes were found in nature in the temperate bacteriophage lambda. Like all temperate bacteriophages, lambda utilizes a lysogenic infection life cycle, wherein its genome is incorporated into the genome of the ''E. coli'' host genome, to excise itself at a later time. Integrase (Int), excisionase (Xis), and integration host factor (IHF) are the enzymes that catalyze the integration and excision of the viral genome, at the previously mentioned ''att'' sites of the viral and host genomes. <br />
<br />
Int cooperatively binds with IHF (which is composed of A and B subunits) in order to catalyze both the integration and excision reactions in the natural lambda phage system shown below. Although int can independently catalyze both reactions, IHF greatly improves int's binding affinity for the att recombination sites by bending the DNA [6]. Although int performs both the forward and reverse reactions, the equilibrium heavily favors the reaction of attB and attP to produce attL and attR sites. Xis binds to the attR sire and serves to shift the equilibrium so that it favors the reverse reaction [5].<br />
<br />
<br />
[[Image:LambdaRecombo.jpg|frame|center|Lambda phage recombination in ''E. coli''. Invitrogen adapted these components from this natural system to produce their Gateway cloning scheme. <br> Image source: http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html]]<br />
<br />
===Invitrogen's adaptation===<br />
<br />
In the Invitrogen scheme, the recombination sites are found in pairs flanking sequences that are intended for transfer. The recombination pairs are directional and specificity is given by ten nucleotides in the core region of each site, as shown below. In addition, the lethal gene ''ccdB'' is incorporated in between the recombination sites in the destination vector to ensure that only the desired recombined product can be cloned.<br />
<br />
[[Image:LR recombination.jpg|frame|center|Directional, site-specific recombination between attL and attR sites. <br> Image source:https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC]]<br />
<br />
The schematic below depicts the LR reaction, during which the attL sites recombine with attR to yield attB and attP sites. The BP reaction proceeds in the opposite direction yielding attL and attR sites.<br />
<br />
[[Image:LR reaction.gif|frame|center|Gateway LR reaction where gene of interest (flanked by attL sites) is transferred to destination vector containing attR sites. <br> Image source:https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC]]<br />
<br />
====Selection for Correct Clones====<br />
<br />
The above scheme employs both ccdB negative selection and antibiotic selection in order to yield >90% of colonies containing the desired expression clone. The entry and destination vectors contain different antibiotic resistances, so plating on the desired antibiotic (Ampicillin in case shown above) eliminates clones containing the entry vector or the by-product of the reaction. In addition, ''ccdB'' is a lethal gene, thereby eliminating colonies containing the destination vector, which would otherwise survive on the antibiotic plate.<br />
<br />
<html><br />
<p align="center"><!-- URL's used in the movie--> <!-- text used in the movie--> <br />
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Animation source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm<br />
<br />
==Gateway ''in vivo''==<br />
<br />
<br />
===Plasmid Based Gateway===<br />
We applied our ideas for facilitating the automation of synthetic biology to the first step of layered assembly--the LR gateway reaction used to move the gene of interest into one or more of the double-antibiotic assembly vectors. We began by attempting to reproduce a plasmid based Gateway reaction, which was similar to that of Invitrogen's Gateway scheme. Although we were successful in having a completely ''in vivo'' version of the Gateway reaction, there were two major drawbacks: we had not eliminated the need for laborious and expensive mini-preps and we had relatively high background from ccdB mutations (the ccdB gene is relatively unstable because of its toxicity).<br />
<br />
We addressed the first drawback by creating a self-lysis device which allows the cell's contents, including the desired product, to be released when arabinose is added. This device was inserted into either the entry or assembly plasmid to facilitate the isolation of the reaction products.<br />
<br />
The second issue was more complex and was resolved by using positive selection methods rather than ccdB negative selection. Efforts to incorporate positive selection for Gateway led to the development of the Genomic Based Gateway scheme.<br />
<br />
===Genomic Based Gateway===<br />
The Genomic Based Gateway scheme involves placement of the assembly vector in the genome. The gene of interest from the entry vector can recombine with the genome to yield the desired product. Both the entry vector and assembly vector have conditional origins of replication which serve as mechanisms for positive selection. Although this is a viable scheme for Gateway and can readily incorporate the lysis device in place of mini-preps, it still requires transformation of the lysate in order to select for the desired product. In an effort to further optimize our Gateway scheme by eliminating the need for transformation, we developed a Phagemid Based Gateway scheme.<br />
<br />
===Phagemid Based Gateway===<br />
The Phagemid Based Gateway utilizes a plasmid that can be packaged in a phage when a lysogenic cell is induced with arabinose. The assembly vector in this scheme contains a phagemid device, which allows the product to be packaged and transferred to another cell without mini-preps or transformation. In addition to simplifying the transfer of the product, the phagemid device serves as a positive selection mechanism and can isolate the desired product when paired with another positive selector, such as an inducible origin of replication.<br />
<br />
==References==<br />
# http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm<br />
# http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html<br />
# http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html|Gateway<br />
# https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC<br />
# Cho, E et al. Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex. ''Journal of Bacteriology''. September 2002; 184(18): 5200–5203. Available Online: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135313 (Accessed: 28 October 2008).<br />
# Frumerie, C et al. Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox. ''Virology''. 5 February 2005; 232(1):284-294. Available Online: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-4F29SN2-1&_user=4420&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=4420&md5=0f9e41ba422140f157a2b1f1fb60b140 (Accessed: 28 October 2008).<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPlasmid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayOverviewTeam:UC Berkeley/GatewayOverview2008-10-30T02:55:51Z<p>Aronlau: /* The Natural Lambda Phage Recombination system */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Gateway Overview'''</font></div><br><br />
==Why Use Gateway?==<br />
<br />
The first step of the layered assembly scheme involves the transfer of biobrick parts from an entry vector to a double antibiotic assembly vector. Traditionally, this would require a fairly work-intensive protocol requiring digestion, gel purification, ligation, transformation, and plasmid isolation. In addition to being more time-consuming, the aforementioned procedure is also suboptimal because it is difficult to scale-up.<br />
<br />
The Gateway Cloning approach developed by [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html Invitrogen] offers a more efficient and convenient alternative for parts transfer. Their procedure involves the enzyme-catalyzed exchange of parts flanked by specific recombination sites. Experimentally, it is a one-pot, room-temperature reaction where the plasmids, buffer, water and enzymes are added together. After the addition of another enzyme and a short incubation period to terminate the reaction, the entire mixture can be transformed directly. This one-pot approach with a fewer steps is much more suitable for large-scale experimentation.<br />
<br />
Gateway is commonly used to facilitate the transfer of a single gene of interest from an entry clone to multiple destination vectors, as shown below. The efficiency and robustness of the Gateway mechanism are ideal for this application because once the gene of interest is cloned and confirmed in the entry vector, subsequent transfers using Gateway need not be confirmed again. Thus, it is ideal for use in the first step of layered assembly where a part may be transferred to one or more of the double antibiotic vectors required for the subsequent assembly steps assembly.<br />
<br />
[[Image:gatewayoverview.png|800px|thumb|center|A entry vector generated from any of the methods can be transferred to various different vectors using the Gateway method. <br>]]<br />
<br />
==Gateway Chemistry==<br />
<br />
===The Natural Lambda Phage Recombination system===<br />
<br />
In general, Gateway reactions in the lab involve the attB, attP, attL, and attR recombination sites and the integrase (Int), excisionase (Xis), and integration host factor (IHF) enzymes. These enzymes were found in nature in the temperate bacteriophage lambda. Like all temperate bacteriophages, lambda utilizes a lysogenic infection life cycle, wherein its genome is incorporated into the genome of the ''E. coli'' host genome, to excise itself at a later time. Integrase (Int), excisionase (Xis), and integration host factor (IHF) are the enzymes that catalyze the integration and excision of the viral genome, at the previously mentioned ''att'' sites of the viral and host genomes. <br />
<br />
Int cooperatively binds with IHF (which is composed of A and B subunits) in order to catalyze both the integration and excision reactions in the natural lambda phage system shown below. Although int can independently catalyze both reactions, IHF greatly improves int's binding affinity for the att recombination sites by bending the DNA [6]. Although int performs both the forward and reverse reactions, the equilibrium heavily favors the reaction of attB and attP to produce attL and attR sites. Xis binds to the attR sire and serves to shift the equilibrium so that it favors the reverse reaction [5].<br />
<br />
<br />
[[Image:LambdaRecombo.jpg|frame|center|10 px|Lambda phage recombination in ''E. coli''. Invitrogen adapted these components from this natural system to produce their Gateway cloning scheme. <br> Image source: http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html]]<br />
<br />
===Invitrogen's adaptation===<br />
<br />
In the Invitrogen scheme, the recombination sites are found in pairs flanking sequences that are intended for transfer. The recombination pairs are directional and specificity is given by ten nucleotides in the core region of each site, as shown below. In addition, the lethal gene ''ccdB'' is incorporated in between the recombination sites in the destination vector to ensure that only the desired recombined product can be cloned.<br />
<br />
[[Image:LR recombination.jpg|frame|center|Directional, site-specific recombination between attL and attR sites. <br> Image source:https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC]]<br />
<br />
The schematic below depicts the LR reaction, during which the attL sites recombine with attR to yield attB and attP sites. The BP reaction proceeds in the opposite direction yielding attL and attR sites.<br />
<br />
[[Image:LR reaction.gif|frame|center|Gateway LR reaction where gene of interest (flanked by attL sites) is transferred to destination vector containing attR sites. <br> Image source:https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC]]<br />
<br />
====Selection for Correct Clones====<br />
<br />
The above scheme employs both ccdB negative selection and antibiotic selection in order to yield >90% of colonies containing the desired expression clone. The entry and destination vectors contain different antibiotic resistances, so plating on the desired antibiotic (Ampicillin in case shown above) eliminates clones containing the entry vector or the by-product of the reaction. In addition, ''ccdB'' is a lethal gene, thereby eliminating colonies containing the destination vector, which would otherwise survive on the antibiotic plate.<br />
<br />
<html><br />
<p align="center"><!-- URL's used in the movie--> <!-- text used in the movie--> <br />
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</p><br />
</html><br />
Animation source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm<br />
<br />
==Gateway ''in vivo''==<br />
<br />
<br />
===Plasmid Based Gateway===<br />
We applied our ideas for facilitating the automation of synthetic biology to the first step of layered assembly--the LR gateway reaction used to move the gene of interest into one or more of the double-antibiotic assembly vectors. We began by attempting to reproduce a plasmid based Gateway reaction, which was similar to that of Invitrogen's Gateway scheme. Although we were successful in having a completely ''in vivo'' version of the Gateway reaction, there were two major drawbacks: we had not eliminated the need for laborious and expensive mini-preps and we had relatively high background from ccdB mutations (the ccdB gene is relatively unstable because of its toxicity).<br />
<br />
We addressed the first drawback by creating a self-lysis device which allows the cell's contents, including the desired product, to be released when arabinose is added. This device was inserted into either the entry or assembly plasmid to facilitate the isolation of the reaction products.<br />
<br />
The second issue was more complex and was resolved by using positive selection methods rather than ccdB negative selection. Efforts to incorporate positive selection for Gateway led to the development of the Genomic Based Gateway scheme.<br />
<br />
===Genomic Based Gateway===<br />
The Genomic Based Gateway scheme involves placement of the assembly vector in the genome. The gene of interest from the entry vector can recombine with the genome to yield the desired product. Both the entry vector and assembly vector have conditional origins of replication which serve as mechanisms for positive selection. Although this is a viable scheme for Gateway and can readily incorporate the lysis device in place of mini-preps, it still requires transformation of the lysate in order to select for the desired product. In an effort to further optimize our Gateway scheme by eliminating the need for transformation, we developed a Phagemid Based Gateway scheme.<br />
<br />
===Phagemid Based Gateway===<br />
The Phagemid Based Gateway utilizes a plasmid that can be packaged in a phage when a lysogenic cell is induced with arabinose. The assembly vector in this scheme contains a phagemid device, which allows the product to be packaged and transferred to another cell without mini-preps or transformation. In addition to simplifying the transfer of the product, the phagemid device serves as a positive selection mechanism and can isolate the desired product when paired with another positive selector, such as an inducible origin of replication.<br />
<br />
==References==<br />
# http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm<br />
# http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html<br />
# http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html|Gateway<br />
# https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC<br />
# Cho, E et al. Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex. ''Journal of Bacteriology''. September 2002; 184(18): 5200–5203. Available Online: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135313 (Accessed: 28 October 2008).<br />
# Frumerie, C et al. Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox. ''Virology''. 5 February 2005; 232(1):284-294. Available Online: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-4F29SN2-1&_user=4420&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=4420&md5=0f9e41ba422140f157a2b1f1fb60b140 (Accessed: 28 October 2008).<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayPlasmid" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:40:56Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|650 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|650 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:37:52Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|600 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|600 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:37:22Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|700 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|700 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:37:09Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|700 px]]<br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|700 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:36:52Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|700 px]] [[Image:generalprocedureGDinEntryVector.png|700 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:36:30Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|600 px]] [[Image:generalprocedureGDinEntryVector.png|600 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:35:44Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|500 px]] [[Image:generalprocedureGDinEntryVector.png|500 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:32:33Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|200 px]] [[Image:generalprocedureGDinEntryVector.png|200 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:29:42Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|200 px]] <br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|200 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:28:58Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|center|200 px]] <br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|center|500 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayPlasmidTeam:UC Berkeley/GatewayPlasmid2008-10-30T02:28:27Z<p>Aronlau: /* General Procedure */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Plasmid Based Gateway'''</font></div><br><br />
<br />
== '''Introduction''' ==<br />
<br />
The current ''in vitro'' LR Gateway procedure for transferring one piece of DNA into another vector without restriction enzymes involves an expensive cocktail of purified plasmids, excisionase, integrase, ihfα and ihfβ. Both an assembly vector (containing the ccdB gene flanked by attR1 and attR2 sites for negative selection) and an entry vector (containing the part(s) desired to be transferred, flanked by attL1 and attL2 sites) are used as the substrates to which the reactants are added. <br><br />
<br />
Inspired by such innovation, we seek to perform this task ''in vivo'' using E. coli to express the reactants necessary. Since E. coli naturally express ihfα/β in their genome, we first tried to integrate excisionase and integrase genes into the genomic DNA as well. However, this proved to be toxic to the cells, making them relatively unstable. Therefore, our next approach was to introduce the reagents into the substrate plasmids. In one case, the the assembly vectors received the Gateway device of the enzymes excisionase and integrase preceded by a temperature sensitive promoter, while in the other case it was introduced into the entry vectors. In both cases, we also explored the effects of additionally introducing the ihfα and ihfβ genes behind the Gateway device. <br><br />
<br />
After careful analysis of our data, we introduced our Lysis device in order to eliminate the mini-prepping procedures and to thus make the entire process cheaper and more efficient.<br><br />
<br />
== '''Gateway Device in Assembly Vector: Plasmid Details '''==<br />
<br />
'''Assembly Vectors:'''Two different variations of the pK112128 assembly vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} between the ccdB site and the attR2 site. These plasmids are named pK112245 and pK112246 respectively (see image links below). A simplified version of the two variants along with the Lysis Device is shown below. <br><br />
[[media:pK112245.png]]<br><br />
[[media:pK112246.png]]<br><br />
[[Image:simpof245or246.png]] <br><br />
<br><br />
<br />
'''Entry Vector:'''The entry vector used in both cases was pBca1256 (see image and link below).<br><br />
[[media:pBca 1256.png]] <br><br />
[[Image:simpof1256.png]] <br><br />
<br><br />
<br />
== '''Gateway Device in Entry Vector: Plasmid Details'''==<br />
<br />
'''Assembly Vector:'''The assembly vector used for each of the following entry vector variations is pK112128 (see link and image below. <br> <br />
[[media:pK112128.png]] <br><br />
[[Image:simpof128.png]] <br><br />
<br><br />
<br />
'''Entry Vectors:''' Two different variations of the pBca1256 entry vector were created: one with {promoter.xis.int!} and one with {promoter.xis.int!}{rbs.ihfα!}{rbs.ihfβ!} just before the attL1 site. These plasmids are named pK112247 and pK112248 respectively (see links below).<br><br />
[[media:pK112247.png]] <br><br />
[[media:pK112248.png]] <br><br />
[[Image:simpof247or248.png]] <br><br />
<br><br />
<br><br />
<br />
== '''General Procedure''' ==<br />
<br />
Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified. <br><br />
<br />
The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below. <br><br />
<br />
[[Image:generalprocedureGDinAssemblyVector.png|500 px]] <br><br />
<br><br />
[[Image:generalprocedureGDinEntryVector.png|500 px]] <br><br />
<br><br />
<br><br />
<br />
== '''Results''' ==<br />
<br />
=== '''Gateway Device in Assembly Vector''' ===<br />
[[Image:plate3.png]] <br><br />
<br><br />
* Control done at 30°C.<br />
[[Image:plate4.png]] <br><br />
<br><br />
* As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.<br />
* In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.<br />
[[Image:plate11d-f.png]] <br><br />
<br><br />
[[Image:plate19d-f.png]] <br><br />
<br><br />
* Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.<br />
<br><br />
'''The Lysis Device'''<br><br />
* The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation. <br />
[[Image:plate245lysis.png]] <br><br />
<br><br />
<br><br />
=== '''Gateway Device in Entry Vector''' ===<br />
[[Image:plate1.png]] <br><br />
<br><br />
[[Image:plate02.png]] <br><br />
* As can been seen above, the Gateway reaction worked with much more efficiency as evidenced by the low rates of cotransformation.<br />
<br><br />
<br />
== '''Materials & Methods''' ==<br />
<br />
We first prepared competent TG1 cells (resistant to ccdB) with the given assembly vector. The assembly vector was transformed into competent TG1 cells and were grown in liquid LB media with Cam/Amp antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 30°C overnight. The following day, 20 ul of the saturated culture was taken out, and was grown under the same conditions to mid-log. About 1 ml of culture was pelleted, the supernatant was discarded and the cells were resuspended in 10 ul KCM plus 90 ul TSS on ice. <br><br />
<br />
<br />
Using these new competent cells, the given entry vector was tranformed into them. They were grown in liquid LB media with Cam/Amp/Spec antibiotics (and 50mM Mg<sup>+2</sup> if the lysis device is included) at 37°C overnight to ensure the cells contain both plasmids and that the temperature sensitive promoter of the gateway device would turn on. A control in which the cells were instead kept at 30°C was conducted to compare our results with those in which the gateway device was theoretically off. <br><br />
<br />
<br />
The following day, the plasmid from about 2 ml of saturated culture was purified. When there was no lysis device in the assembly vector, plasmid purification was achieved with the QIAprep Spin Miniprep Kit. In the case where the lysis device is integrated into the assembly plasmid, the culture was pelleted, the supernatant removed, resuspended in 1 ml PBS, pelleted, the supernatant removed, and finally resuspended again in 100 ul PBS. <br><br />
<br />
<br />
The purified plasmids were next transformed into MG1061 cells (sensitive to ccdB). These were plated on LB agar plates with Cam/Amp antibiotics and grown overnight at 37°C. The next day, 16 colonies were picked from each plate, spotted on a Cam/Amp antibiotic plate, as well as Spec antibiotic plate to test for cotransformance of the entry vector with the desired product. <br><br />
<br />
<br />
The above procedure were conducted in parallel with different combinations of assembly and entry vectors. The following combinations of assembly and entry vectors were done: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Each combination included a control in which the Gateway device was not turned on during the allotted time for the reaction to occur (i.e. they were grown at 30°C, when the temperature sensitive promoter is off). In addition to this, three trials of each were conducted. Finally, in order to see if gateway could be performed on parts of different sizes, five different parts in pBca1256 were chosen (Bca1117, Bca1133, Bca1270, Bca1252, and Bca1168) and tested with the assembly vector pK112245, as well as with pK112245. One colony from each was sequenced to ensure gateway had occurred by identification of attB1 and attB2 sites as well as the parts in the assembly vectors. <br><br />
<br />
<br />
After data analysis, we suspected that higher efficiency could be achieved for the schemes in which the Gateway Device was in the assembly vector if less plasmid was used to transform into MG1061 cells. Therefore, we took the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256 and diluted each 20 times. Three trials for each in which the 20x dilution of the purified plasmid was transformed into MG1061 cells was performed under the same conditions, and 32 colonies from each plate were picked the following day. <br><br />
<br />
<br />
Other controls were also performed. Each assembly vector used was also transformed into into MG1061 cells which were put on Cam/Amp antibiotic LB agar plates to make sure ccdB was sufficient for negative selection. Only a very few number of white colonies grew for each, which was expected since it is known that ccdB is prone to mutation and weakening. The entry vectors were also each individually transformed into MC1061 cells and plated on Cam/Amp antibiotic plates. No colonies appeared, proving that the entry vector did not contain Cam/Amp resistance. <br><br />
<br />
== '''Discussion''' ==<br />
<br />
* Our desired product is a reacted assembly vector with the entry vector mRFP (or part) between attB1 and attB2 sites. Since the origin of replication in the assembly vectors is low-copy, the cells should appear light pink in color.<br />
<br />
* Colonies that are bright pink, which grow on both Cam/Amp and Spec antibiotic plates, are cotransformed. This means that in addition to our desired assembly vector-with-part plasmid, one of the original, unreacted entry vectors has entered into the cell. These appear as bright pink because the origin of replication in the entry vector is high-copy.<br />
<br />
* White colonies first appeared to be evidence of a mutation in the ccdB gene such that it inhibits its toxicity. Since ccdB is used to negatively select against the original, unreacted assembly vector containing ccdB flanked by attR1 and attR2, a mutation which weakens ccdB such that a strain sensitive to normal ccdB can grow in spite of the gene's presence and gives undesirable background products. However, after sequencing 4 different white colonies, we discovered that in each case the mRFP gene was indeed inserted into the assembly vector and was flanked by attB1 and attB2 sites (our desired product); the only problem was that there were deletions (24 or 25 bp in length) in the promoter of the part which was driving the expression of mRFP! Thus, the few white colonies seen are likely due to mutations in the original part rather than a mutation in ccdB.<br />
<br />
* There are also some colonies that are very slightly pink. We predict that these are cells which contain the desired assembly vector-with-part plasmid, but that the part itself has sustained some sort of mutation (perhaps again in the promoter) that slightly weakens the expression of mRFP.<br />
<br />
* Proportionally, the less background we receive at the end of the experiment, the more efficient the plasmid based Gateway reaction is.<br />
<br />
* From our experimental results comparing the "Gateway Device" with and without ihfα/β, we conclude that the additional expression of ihfα/β is unnecessary and makes no difference in the plasmid based gateway reactions. However, based on the difficulty of previous attempts to join and clone ihfα and ihfβ, it has been concluded that these parts are unnecessary or even hazardous to cells, and thus will not be explored further in other Gateway schemes.<br />
<br />
* Our data suggests that our procedure was most efficient when the Gateway Device was located in the entry vectors as opposed to the assembly vectors. This makes sense since the entry vector origin of replication is high copy, meaning the reagents necessary for the reaction to occur (excisionase, integrase, ihfα and ihfβ) will be expressed at much higher levels when the temperature sensitive promoter is turned on. More reagents produced increases the probability that the Gateway reaction will occur within a given cell, and that we will get our desired product with the greatest efficiency. However, from experience in creating and cloning the Gateway Device, we know that this composite part is somewhat toxic to the cell since there are att sites in the genomic DNA of E. coli. Thus, it is necessary to carefully control this device with, in this case, a temperature sensitive promoter. This also means that if the "part" in your entry vector is also somewhat toxic, the additional toxicity of the Gateway Device may make the entry vector difficult or even impossible to make in the first place.<br />
<br />
* Therefore, it is most beneficial to put the Gateway Device into the assembly vector. In order to improve the efficiency and reduce the background in these cases, we diluted the post-reaction purified plasmid mixtures from the trials involving pK112245 + pBca1256 and pK112246 + pBca1256. Our results show a significant reduction in cotransformation and thus a higher rate of efficiency.<br />
<br />
* The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.<br />
<br />
* All in all, we were able to successfully conduct the Gateway reaction ''in vivo'' using assembly and entry plasmids with relatively high efficiency. Our problems with cotransformation seem to largely reduced upon diluting the purified plasmid before transformation. The white colony background due to mutated, weakened ccdB may be solved by replacing it with or adding on another more robust toxic protein, such as barnase, to aid in negative selection. Another possibility would be to use positive selection, for example by using conditional replication of an origin.<br />
<br />
* Although the plasmid based Gateway scheme has proven to be successful, we have also tried other methods to achieve the Gateway reaction: see Phagemid Based Gateway and Genomic Based Gateway.<br />
<br />
<html><br />
<a href="https://2008.igem.org/Team:UC_Berkeley/GatewayGenomic" class="titleIcon"><br />
<img align=right src="https://static.igem.org/mediawiki/2008/9/9a/GreyNext.png"><br />
</a><br />
</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayOverviewTeam:UC Berkeley/GatewayOverview2008-10-30T02:27:48Z<p>Aronlau: /* Invitrogen's adaptation */</p>
<hr />
<div>__NOTOC__<br />
{{UCBmain|cssLink=}}<br />
<div style="text-align: center;"><font size="6">'''Gateway Overview'''</font></div><br><br />
==Why Use Gateway?==<br />
<br />
The first step of the layered assembly scheme involves the transfer of biobrick parts from an entry vector to a double antibiotic assembly vector. Traditionally, this would require a fairly work-intensive protocol requiring digestion, gel purification, ligation, transformation, and plasmid isolation. In addition to being more time-consuming, the aforementioned procedure is also suboptimal because it is difficult to scale-up.<br />
<br />
The Gateway Cloning approach developed by [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html Invitrogen] offers a more efficient and convenient alternative for parts transfer. Their procedure involves the enzyme-catalyzed exchange of parts flanked by specific recombination sites. Experimentally, it is a one-pot, room-temperature reaction where the plasmids, buffer, water and enzymes are added together. After the addition of another enzyme and a short incubation period to terminate the reaction, the entire mixture can be transformed directly. This one-pot approach with a fewer steps is much more suitable for large-scale experimentation.<br />
<br />
Gateway is commonly used to facilitate the transfer of a single gene of interest from an entry clone to multiple destination vectors, as shown below. The efficiency and robustness of the Gateway mechanism are ideal for this application because once the gene of interest is cloned and confirmed in the entry vector, subsequent transfers using Gateway need not be confirmed again. Thus, it is ideal for use in the first step of layered assembly where a part may be transferred to one or more of the double antibiotic vectors required for the subsequent assembly steps assembly.<br />
<br />
[[Image:gatewayoverview.png|800px|thumb|center|A entry vector generated from any of the methods can be transferred to various different vectors using the Gateway method. <br>]]<br />
<br />
==Gateway Chemistry==<br />
<br />
===The Natural Lambda Phage Recombination system===<br />
<br />
In general, Gateway reactions in the lab involve the attB, attP, attL, and attR recombination sites and the integrase (Int), excisionase (Xis), and integration host factor (IHF) enzymes. These enzymes were found in nature in the temperate bacteriophage lambda. Like all temperate bacteriophages, lambda utilizes a lysogenic infection life cycle, wherein its genome is incorporated into the genome of the ''E. coli'' host genome, to excise itself at a later time. Integrase (Int), excisionase (Xis), and integration host factor (IHF) are the enzymes that catalyze the integration and excision of the viral genome, at the previously mentioned ''att'' sites of the viral and host genomes. <br />
<br />
Int cooperatively binds with IHF (which is composed of A and B subunits) in order to catalyze both the integration and excision reactions in the natural lambda phage system shown below. Although int can independently catalyze both reactions, IHF greatly improves int's binding affinity for the att recombination sites by bending the DNA [6]. Although int performs both the forward and reverse reactions, the equilibrium heavily favors the reaction of attB and attP to produce attL and attR sites. Xis binds to the attR sire and serves to shift the equilibrium so that it favors the reverse reaction [5].<br />
<br />
<br />
[[Image:LambdaRecombination.jpg|frame|center|Lambda phage recombination in ''E. coli''. Invitrogen adapted these components from this natural system to produce their Gateway cloning scheme. <br> Image source: http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html]]<br />
<br />
===Invitrogen's adaptation===<br />
<br />
In the Invitrogen scheme, the recombination sites are found in pairs flanking sequences that are intended for transfer. The recombination pairs are directional and specificity is given by ten nucleotides in the core region of each site, as shown below. In addition, the lethal gene ''ccdB'' is incorporated in between the recombination sites in the destination vector to ensure that only the desired recombined product can be cloned.<br />
<br />
[[Image:LR recombination.jpg|frame|center|Directional, site-specific recombination between attL and attR sites. <br> Image source:https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC]]<br />
<br />
The schematic below depicts the LR reaction, during which the attL sites recombine with attR to yield attB and attP sites. The BP reaction proceeds in the opposite direction yielding attL and attR sites.<br />
<br />
[[Image:LR reaction.gif|frame|center|Gateway LR reaction where gene of interest (flanked by attL sites) is transferred to destination vector containing attR sites. <br> Image source:https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC]]<br />
<br />
====Selection for Correct Clones====<br />
<br />
The above scheme employs both ccdB negative selection and antibiotic selection in order to yield >90% of colonies containing the desired expression clone. The entry and destination vectors contain different antibiotic resistances, so plating on the desired antibiotic (Ampicillin in case shown above) eliminates clones containing the entry vector or the by-product of the reaction. In addition, ''ccdB'' is a lethal gene, thereby eliminating colonies containing the destination vector, which would otherwise survive on the antibiotic plate.<br />
<br />
<html><br />
<p align="center"><!-- URL's used in the movie--> <!-- text used in the movie--> <br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://active.macromedia.com/flash2/cabs/swflash.cab#version=4,0,0,0" ID="cloning" WIDTH="550" HEIGHT="400"><br />
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Animation source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm<br />
<br />
==Gateway ''in vivo''==<br />
<br />
<br />
===Plasmid Based Gateway===<br />
We applied our ideas for facilitating the automation of synthetic biology to the first step of layered assembly--the LR gateway reaction used to move the gene of interest into one or more of the double-antibiotic assembly vectors. We began by attempting to reproduce a plasmid based Gateway reaction, which was similar to that of Invitrogen's Gateway scheme. Although we were successful in having a completely ''in vivo'' version of the Gateway reaction, there were two major drawbacks: we had not eliminated the need for laborious and expensive mini-preps and we had relatively high background from ccdB mutations (the ccdB gene is relatively unstable because of its toxicity).<br />
<br />
We addressed the first drawback by creating a self-lysis device which allows the cell's contents, including the desired product, to be released when arabinose is added. This device was inserted into either the entry or assembly plasmid to facilitate the isolation of the reaction products.<br />
<br />
The second issue was more complex and was resolved by using positive selection methods rather than ccdB negative selection. Efforts to incorporate positive selection for Gateway led to the development of the Genomic Based Gateway scheme.<br />
<br />
===Genomic Based Gateway===<br />
The Genomic Based Gateway scheme involves placement of the assembly vector in the genome. The gene of interest from the entry vector can recombine with the genome to yield the desired product. Both the entry vector and assembly vector have conditional origins of replication which serve as mechanisms for positive selection. Although this is a viable scheme for Gateway and can readily incorporate the lysis device in place of mini-preps, it still requires transformation of the lysate in order to select for the desired product. In an effort to further optimize our Gateway scheme by eliminating the need for transformation, we developed a Phagemid Based Gateway scheme.<br />
<br />
===Phagemid Based Gateway===<br />
The Phagemid Based Gateway utilizes a plasmid that can be packaged in a phage when a lysogenic cell is induced with arabinose. The assembly vector in this scheme contains a phagemid device, which allows the product to be packaged and transferred to another cell without mini-preps or transformation. In addition to simplifying the transfer of the product, the phagemid device serves as a positive selection mechanism and can isolate the desired product when paired with another positive selector, such as an inducible origin of replication.<br />
<br />
==References==<br />
# http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm<br />
# http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html<br />
# http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html|Gateway<br />
# https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC<br />
# Cho, E et al. Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex. ''Journal of Bacteriology''. September 2002; 184(18): 5200–5203. Available Online: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135313 (Accessed: 28 October 2008).<br />
# Frumerie, C et al. Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox. ''Virology''. 5 February 2005; 232(1):284-294. Available Online: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-4F29SN2-1&_user=4420&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=4420&md5=0f9e41ba422140f157a2b1f1fb60b140 (Accessed: 28 October 2008).<br />
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</html></div>Aronlauhttp://2008.igem.org/Team:UC_Berkeley/GatewayOverviewTeam:UC Berkeley/GatewayOverview2008-10-30T02:27:34Z<p>Aronlau: /* Invitrogen's adaptation */</p>
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<div style="text-align: center;"><font size="6">'''Gateway Overview'''</font></div><br><br />
==Why Use Gateway?==<br />
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The first step of the layered assembly scheme involves the transfer of biobrick parts from an entry vector to a double antibiotic assembly vector. Traditionally, this would require a fairly work-intensive protocol requiring digestion, gel purification, ligation, transformation, and plasmid isolation. In addition to being more time-consuming, the aforementioned procedure is also suboptimal because it is difficult to scale-up.<br />
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The Gateway Cloning approach developed by [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html Invitrogen] offers a more efficient and convenient alternative for parts transfer. Their procedure involves the enzyme-catalyzed exchange of parts flanked by specific recombination sites. Experimentally, it is a one-pot, room-temperature reaction where the plasmids, buffer, water and enzymes are added together. After the addition of another enzyme and a short incubation period to terminate the reaction, the entire mixture can be transformed directly. This one-pot approach with a fewer steps is much more suitable for large-scale experimentation.<br />
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Gateway is commonly used to facilitate the transfer of a single gene of interest from an entry clone to multiple destination vectors, as shown below. The efficiency and robustness of the Gateway mechanism are ideal for this application because once the gene of interest is cloned and confirmed in the entry vector, subsequent transfers using Gateway need not be confirmed again. Thus, it is ideal for use in the first step of layered assembly where a part may be transferred to one or more of the double antibiotic vectors required for the subsequent assembly steps assembly.<br />
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[[Image:gatewayoverview.png|800px|thumb|center|A entry vector generated from any of the methods can be transferred to various different vectors using the Gateway method. <br>]]<br />
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==Gateway Chemistry==<br />
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===The Natural Lambda Phage Recombination system===<br />
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In general, Gateway reactions in the lab involve the attB, attP, attL, and attR recombination sites and the integrase (Int), excisionase (Xis), and integration host factor (IHF) enzymes. These enzymes were found in nature in the temperate bacteriophage lambda. Like all temperate bacteriophages, lambda utilizes a lysogenic infection life cycle, wherein its genome is incorporated into the genome of the ''E. coli'' host genome, to excise itself at a later time. Integrase (Int), excisionase (Xis), and integration host factor (IHF) are the enzymes that catalyze the integration and excision of the viral genome, at the previously mentioned ''att'' sites of the viral and host genomes. <br />
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Int cooperatively binds with IHF (which is composed of A and B subunits) in order to catalyze both the integration and excision reactions in the natural lambda phage system shown below. Although int can independently catalyze both reactions, IHF greatly improves int's binding affinity for the att recombination sites by bending the DNA [6]. Although int performs both the forward and reverse reactions, the equilibrium heavily favors the reaction of attB and attP to produce attL and attR sites. Xis binds to the attR sire and serves to shift the equilibrium so that it favors the reverse reaction [5].<br />
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[[Image:LambdaRecombination.jpg|frame|center|Lambda phage recombination in ''E. coli''. Invitrogen adapted these components from this natural system to produce their Gateway cloning scheme. <br> Image source: http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html]]<br />
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===Invitrogen's adaptation===<br />
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In the Invitrogen scheme, the recombination sites are found in pairs flanking sequences that are intended for transfer. The recombination pairs are directional and specificity is given by ten nucleotides in the core region of each site, as shown below. In addition, the lethal gene ''ccdB'' is incorporated in between the recombination sites in the destination vector to ensure that only the desired recombined product can be cloned.<br />
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[[Image:LR recombination.jpg|frame|center|Directional, site-specific recombination between attL and attR sites. <br> Image source:https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC]]<br />
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The schematic below depicts the LR reaction, during which the attL sites recombine with attR to yield attB and attP sites. The BP reaction proceeds in the opposite direction yielding attL and attR sites.<br />
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[[Image:LR reaction.gif|frame|center|Gateway LR reaction where gene of interest (flanked by attL sites) is transferred to destination vector containing attR sites. <br> Image sourcehttps://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC]]<br />
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====Selection for Correct Clones====<br />
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The above scheme employs both ccdB negative selection and antibiotic selection in order to yield >90% of colonies containing the desired expression clone. The entry and destination vectors contain different antibiotic resistances, so plating on the desired antibiotic (Ampicillin in case shown above) eliminates clones containing the entry vector or the by-product of the reaction. In addition, ''ccdB'' is a lethal gene, thereby eliminating colonies containing the destination vector, which would otherwise survive on the antibiotic plate.<br />
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Animation source: http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm<br />
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==Gateway ''in vivo''==<br />
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===Plasmid Based Gateway===<br />
We applied our ideas for facilitating the automation of synthetic biology to the first step of layered assembly--the LR gateway reaction used to move the gene of interest into one or more of the double-antibiotic assembly vectors. We began by attempting to reproduce a plasmid based Gateway reaction, which was similar to that of Invitrogen's Gateway scheme. Although we were successful in having a completely ''in vivo'' version of the Gateway reaction, there were two major drawbacks: we had not eliminated the need for laborious and expensive mini-preps and we had relatively high background from ccdB mutations (the ccdB gene is relatively unstable because of its toxicity).<br />
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We addressed the first drawback by creating a self-lysis device which allows the cell's contents, including the desired product, to be released when arabinose is added. This device was inserted into either the entry or assembly plasmid to facilitate the isolation of the reaction products.<br />
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The second issue was more complex and was resolved by using positive selection methods rather than ccdB negative selection. Efforts to incorporate positive selection for Gateway led to the development of the Genomic Based Gateway scheme.<br />
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===Genomic Based Gateway===<br />
The Genomic Based Gateway scheme involves placement of the assembly vector in the genome. The gene of interest from the entry vector can recombine with the genome to yield the desired product. Both the entry vector and assembly vector have conditional origins of replication which serve as mechanisms for positive selection. Although this is a viable scheme for Gateway and can readily incorporate the lysis device in place of mini-preps, it still requires transformation of the lysate in order to select for the desired product. In an effort to further optimize our Gateway scheme by eliminating the need for transformation, we developed a Phagemid Based Gateway scheme.<br />
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===Phagemid Based Gateway===<br />
The Phagemid Based Gateway utilizes a plasmid that can be packaged in a phage when a lysogenic cell is induced with arabinose. The assembly vector in this scheme contains a phagemid device, which allows the product to be packaged and transferred to another cell without mini-preps or transformation. In addition to simplifying the transfer of the product, the phagemid device serves as a positive selection mechanism and can isolate the desired product when paired with another positive selector, such as an inducible origin of replication.<br />
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==References==<br />
# http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring2000/patton/gateway.htm<br />
# http://tools.invitrogen.com/downloads/gateway-the-basics-seminar.html<br />
# http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html|Gateway<br />
# https://commerce.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=290D0521B5FFA1B782C62C0AB62FD7BC<br />
# Cho, E et al. Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex. ''Journal of Bacteriology''. September 2002; 184(18): 5200–5203. Available Online: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=135313 (Accessed: 28 October 2008).<br />
# Frumerie, C et al. Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox. ''Virology''. 5 February 2005; 232(1):284-294. Available Online: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-4F29SN2-1&_user=4420&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=4420&md5=0f9e41ba422140f157a2b1f1fb60b140 (Accessed: 28 October 2008).<br />
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</html></div>Aronlau