Team:BrownTwo/Protocols/bbyeastchrom

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    <strong>                </strong>The Registry  includes a growing number of parts for use in yeast, but there currently exist  no BBa compatible yeast shuttle vectors.  In our work, we have chosen to utilize Sikorski yeast vectors to  integrate BBa parts into yeast chromosomes.  [1]  With genomic insertion, each  transformation is stable, being reproduced with the rest of the  chromosome.  Additionally, exactly one  copy of each insertion is present in transformed strains, allowing for precise  control of the relative expression levels of multiple elements.  The Sikorski vectors can be transformed into  E. coli for construction and propagation.  There exist four integrative vectors in the pRS30x family, each  integrating at a different auxotrophic locus and containing the amino acid  synthesis gene to fill that auxotrophy as a marker for integration.  Unfortunately, the vectors are not Biobrick compatable- they each contain one extraneous Biobrick site, and lack the standard cloning site.  If time permits,  we would like to make these vectors BBa compatible.  In the meantime, there do exist simple  methods for cloning BBa parts onto the pRS30x vectors for yeast chromosomal  integration[2] We have taken a cue  from Pam Silver’s lab in using the following protocols: <br />
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<p>The Registry  includes a growing number of parts for use in yeast, but there currently exist  no BBa compatible yeast shuttle vectors.  In our work, we have chosen to utilize Sikorski yeast vectors to  integrate BBa parts into yeast chromosomes.  [1]  With genomic insertion, each  transformation is stable, being reproduced with the rest of the  chromosome.  Additionally, exactly one  copy of each insertion is present in transformed strains, allowing for precise  control of the relative expression levels of multiple elements.  The Sikorski vectors can be transformed into  E. coli for construction and propagation.  There exist four integrative vectors in the pRS30x family, each  integrating at a different auxotrophic locus and containing the amino acid  synthesis gene to fill that auxotrophy as a marker for integration.  The vectors are not suitable for Biobrick construction- they each contain one extraneous Biobrick site, and lack the standard cloning site.  Thankfully, there is a standard method for cloning BBa parts onto the pRS30x vectors for yeast chromosomal  integration: [2]  </p><br />
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    <em>Shuttling BBa constructs onto yeast chromosomes</em></p>
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   <li>Digest the BBa vector containing the finished part and pRS30x vector of choice using the following enzymes:</li>
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   <li>Digest the BBa vector containing the finished construct and pRS30x vector of choice using the following enzymes:</li><br>
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<p>Alternatively,  the final BBa construction ligation can be done as a double insertion onto a  pRS30x vector:</p>
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<p>Alternatively,  the final BBa construction ligation can be done as a double insertion onto a  pRS30x vector.  We did all of our pRS vector insertions this way.  The three-way ligations work well consistently, and many variations (e.g. combination of promoters and coding regions) can be generated with relative ease:</p>
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   <li> Heat-kill and/or purify the digest</li>
   <li> Heat-kill and/or purify the digest</li>
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   <li>Perform yeast transformation  using a standard lithium acetate procedure.  We have gotten good results from the following:</li>
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   <li>Transform the linear construct into a haploid strain with the correct auxotrophies (His, Trp, Leu, Ura- we used W303) using a standard lithium acetate procedure.  We have gotten good results from the following:</li>
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<p><a href="http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php">http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php</a></p>
<p><a href="http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php">http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php</a></p>
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Select for transformants by plating the transformation on a synthetic defined (SD) plate with amino acid dropouts for whatever gene is encoded on the vector you transformed.  Double integrations (integrating two constructs at ones) worked very reliably for us, and triple integrations worked about half the time.
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If you need to integrate more than four constructs, you can transform up to three into each haploid mating type (a and alpha), then mate them to yield a diploid strain with all of your constructs.  Use complementary selection markers (e.g. His, Trp, Leu in type a and Trp, Leu, Ura in type alpha) so you can plate the mixed cells on SD-His-Trp-Leu-Ura and select for cells that have mated, and thus have all of your constructs in their genome.
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<p>[1] <a href="http://www.genetics.org/cgi/reprint/122/1/19.pdf" title="http://www.genetics.org/cgi/reprint/122/1/19.pdf">http://www.genetics.org/cgi/reprint/122/1/19.pdf</a><br />
<p>[1] <a href="http://www.genetics.org/cgi/reprint/122/1/19.pdf" title="http://www.genetics.org/cgi/reprint/122/1/19.pdf">http://www.genetics.org/cgi/reprint/122/1/19.pdf</a><br />
   [2]  <a href="http://openwetware.org/wiki/Silver:_BB_Strategy">http://openwetware.org/wiki/Silver:_BB_Strategy</a></p>
   [2]  <a href="http://openwetware.org/wiki/Silver:_BB_Strategy">http://openwetware.org/wiki/Silver:_BB_Strategy</a></p>
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Latest revision as of 04:42, 30 October 2008



Moving Biobrick Constructs onto Yeast Chromosomes

A New Standard for Biobrick Construction


The Registry includes a growing number of parts for use in yeast, but there currently exist no BBa compatible yeast shuttle vectors. In our work, we have chosen to utilize Sikorski yeast vectors to integrate BBa parts into yeast chromosomes. [1] With genomic insertion, each transformation is stable, being reproduced with the rest of the chromosome. Additionally, exactly one copy of each insertion is present in transformed strains, allowing for precise control of the relative expression levels of multiple elements. The Sikorski vectors can be transformed into E. coli for construction and propagation. There exist four integrative vectors in the pRS30x family, each integrating at a different auxotrophic locus and containing the amino acid synthesis gene to fill that auxotrophy as a marker for integration. The vectors are not suitable for Biobrick construction- they each contain one extraneous Biobrick site, and lack the standard cloning site. Thankfully, there is a standard method for cloning BBa parts onto the pRS30x vectors for yeast chromosomal integration: [2]


  • Digest the BBa vector containing the finished construct and pRS30x vector of choice using the following enzymes:

Sikorski vector (marker)

insert & vector digest

pRS303 (HIS3)

EcoRI, SpeI

pRS304* (TRP1)

EcoRI, SpeI

pRS305 (LEU2)

XbaI, PstI

pRS306 (URA3)

EcoRI, SpeI


Alternatively, the final BBa construction ligation can be done as a double insertion onto a pRS30x vector. We did all of our pRS vector insertions this way. The three-way ligations work well consistently, and many variations (e.g. combination of promoters and coding regions) can be generated with relative ease:


Sikorski vector

vector digest

forward part digest

back part digest

pRS303 (HIS3)

EcoRI, Not I

EcoRI, SpeI

XbaI, NotI

pRS304* (TRP1)

EcoRI, Not I

EcoRI, SpeI

XbaI, NotI

pRS305 (LEU2)

NotI, SpeI

NotI, PstI

XbaI, PstI

pRS306 (URA3)

EcoRI, Not I

EcoRI, SpeI

XbaI, NotI

    • (Note: The orientation of the BioBrick part should be opposite that of the auxotropic gene once incorporated into the Sikorski vector.)
  • Ligate the insert and vector using standard techniques and transform into E. coli
  • Miniprep the vector from the transformed strain
  • Linearize ~1 microgram of the vector per transformation using the appropriate enzyme according to the following table:

 

Vector

Marker

Linearization Enzyme

pRS303

HIS3

PstI

pRS304*

TRP1

PstI

pRS305

LEU2

BstEII

pRS306

URA3

PstI

  • Heat-kill and/or purify the digest
  • Transform the linear construct into a haploid strain with the correct auxotrophies (His, Trp, Leu, Ura- we used W303) using a standard lithium acetate procedure. We have gotten good results from the following:

http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php

Select for transformants by plating the transformation on a synthetic defined (SD) plate with amino acid dropouts for whatever gene is encoded on the vector you transformed. Double integrations (integrating two constructs at ones) worked very reliably for us, and triple integrations worked about half the time. If you need to integrate more than four constructs, you can transform up to three into each haploid mating type (a and alpha), then mate them to yield a diploid strain with all of your constructs. Use complementary selection markers (e.g. His, Trp, Leu in type a and Trp, Leu, Ura in type alpha) so you can plate the mixed cells on SD-His-Trp-Leu-Ura and select for cells that have mated, and thus have all of your constructs in their genome.

[1] http://www.genetics.org/cgi/reprint/122/1/19.pdf
[2] http://openwetware.org/wiki/Silver:_BB_Strategy