Team:BrownTwo/Implementation/construction

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=Construction=
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=Biobrick Construction in Yeast=
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==Modular Transcription Factors==
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<html>
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<p><strong>Moving Biobrick Parts  onto Yeast Chromosomes</strong><br />
 +
    <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 />
 +
    <em>Shuttling BBa constructs onto yeast chromosomes</em></p>
 +
<ul>
 +
  <li>Digest the BBa vector containing the finished  part and pRS30x vector of choice using the following enzymes:</li>
 +
</ul>
 +
<div align="center">
 +
  <table border="1" cellpadding="0">
 +
    <tr>
 +
      <td><p>Sikorski    vector (marker)</p></td>
 +
      <td><p>insert &amp;    vector digest </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td><p>pRS303 (HIS3) </p></td>
 +
      <td><p>EcoRI, SpeI </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td><p>pRS304* (TRP1) </p></td>
 +
      <td><p>EcoRI, SpeI </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td><p>pRS305 (LEU2) </p></td>
 +
      <td><p>XbaI, PstI</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td><p>pRS306 (URA3) </p></td>
 +
      <td><p>EcoRI, SpeI </p></td>
 +
    </tr>
 +
  </table>
 +
</div>
 +
<p>Alternatively,  the final BBa construction ligation can be done as a double insertion onto a  pRS30x vector:</p>
 +
<div align="center">
 +
  <table border="1" cellpadding="0">
 +
    <tr>
 +
      <td><br />
 +
        Sikorski    vector </td>
 +
      <td><p>vector digest </p></td>
 +
      <td><p>forward part    digest </p></td>
 +
      <td><p>back part    digest </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td><p>pRS303 (HIS3)</p></td>
 +
      <td><p>EcoRI, Not I </p></td>
 +
      <td><p>EcoRI, SpeI</p></td>
 +
      <td><p>XbaI, NotI</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td><p>pRS304*    (TRP1) </p></td>
 +
      <td><p>EcoRI, Not I </p></td>
 +
      <td><p>EcoRI, SpeI</p></td>
 +
      <td><p>XbaI, NotI</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td><p>pRS305 (LEU2) </p></td>
 +
      <td><p>NotI, SpeI</p></td>
 +
      <td><p>NotI, PstI</p></td>
 +
      <td><p>XbaI, PstI</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td><p>pRS306 (URA3) </p></td>
 +
      <td><p>EcoRI, Not I </p></td>
 +
      <td><p>EcoRI, SpeI</p></td>
 +
      <td><p>XbaI, NotI</p></td>
 +
    </tr>
 +
  </table>
 +
</div>
 +
<ul type="disc">
 +
  <ul type="circle">
 +
    <li>(Note: The orientation        of the BioBrick part should be opposite that of the auxotropic gene once        incorporated into the Sikorski vector.)</li>
 +
  </ul>
 +
</ul>
 +
<ul>
 +
  <li>Ligate the insert and vector  using standard techniques and transform into E. coli</li>
 +
  <li>Miniprep the vector from the  transformed strain</li>
 +
  <li>Linearize ~1 microgram of the  vector per transformation using the appropriate enzyme according to the  following table:</li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<table border="1" cellspacing="0" cellpadding="0">
 +
  <tr>
 +
    <td width="213" valign="top"><p>Vector</p></td>
 +
    <td width="213" valign="top"><p>Marker</p></td>
 +
    <td width="213" valign="top"><p>Linearization    Enzyme</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="213" valign="top"><p>pRS303</p></td>
 +
    <td width="213" valign="top"><p>HIS3</p></td>
 +
    <td width="213" valign="top"><p>PstI</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="213" valign="top"><p>pRS304*</p></td>
 +
    <td width="213" valign="top"><p>TRP1</p></td>
 +
    <td width="213" valign="top"><p>PstI</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="213" valign="top"><p>pRS305</p></td>
 +
    <td width="213" valign="top"><p>LEU2</p></td>
 +
    <td width="213" valign="top"><p>BstEII</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="213" valign="top"><p>pRS306</p></td>
 +
    <td width="213" valign="top"><p>URA3</p></td>
 +
    <td width="213" valign="top"><p>PstI</p></td>
 +
  </tr>
 +
</table>
 +
<ul>
 +
  <li> Heat-kill and/or purify the digest</li>
 +
  <li>Perform yeast transformation  using a standard lithium acetate procedure.   We have gotten good results from the following:</li>
 +
</ul>
 +
<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>[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>
 +
 
 +
</html>

Revision as of 02:37, 30 October 2008



Biobrick Construction in Yeast

Moving Biobrick Parts onto Yeast Chromosomes
                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: 
Shuttling BBa constructs onto yeast chromosomes

  • Digest the BBa vector containing the finished part 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:


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
  • Perform yeast transformation 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

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