Team:BrownTwo/Implementation/construction

From 2008.igem.org

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==Biobrick Construction in Yeast==
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==A Modular Approach to Constructing the Circuit==
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[[image:Limiter_diagram.PNG|center|600px]]
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<p><strong>Moving Biobrick Parts  onto Yeast Chromosomes</strong><br />
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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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    <em>Shuttling BBa constructs onto yeast chromosomes</em></p>
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<ul>
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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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</ul>
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<div align="center">
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  <table border="1" cellpadding="0">
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    <tr>
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      <td><p>Sikorski    vector (marker)</p></td>
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      <td><p>insert &amp;    vector digest </p></td>
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    </tr>
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    <tr>
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      <td><p>pRS303 (HIS3) </p></td>
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      <td><p>EcoRI, SpeI </p></td>
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    </tr>
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    <tr>
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      <td><p>pRS304* (TRP1) </p></td>
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      <td><p>EcoRI, SpeI </p></td>
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    </tr>
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    <tr>
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      <td><p>pRS305 (LEU2) </p></td>
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      <td><p>XbaI, PstI</p></td>
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    </tr>
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    <tr>
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      <td><p>pRS306 (URA3) </p></td>
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      <td><p>EcoRI, SpeI </p></td>
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    </tr>
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  </table>
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</div>
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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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<div align="center">
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  <table border="1" cellpadding="0">
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    <tr>
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      <td><br />
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        Sikorski    vector </td>
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      <td><p>vector digest </p></td>
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      <td><p>forward part    digest </p></td>
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      <td><p>back part    digest </p></td>
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    </tr>
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    <tr>
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      <td><p>pRS303 (HIS3)</p></td>
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      <td><p>EcoRI, Not I </p></td>
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      <td><p>EcoRI, SpeI</p></td>
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      <td><p>XbaI, NotI</p></td>
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    </tr>
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    <tr>
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      <td><p>pRS304*    (TRP1) </p></td>
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      <td><p>EcoRI, Not I </p></td>
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      <td><p>EcoRI, SpeI</p></td>
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      <td><p>XbaI, NotI</p></td>
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    </tr>
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    <tr>
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      <td><p>pRS305 (LEU2) </p></td>
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      <td><p>NotI, SpeI</p></td>
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      <td><p>NotI, PstI</p></td>
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      <td><p>XbaI, PstI</p></td>
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    </tr>
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    <tr>
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      <td><p>pRS306 (URA3) </p></td>
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      <td><p>EcoRI, Not I </p></td>
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      <td><p>EcoRI, SpeI</p></td>
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      <td><p>XbaI, NotI</p></td>
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    </tr>
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  </table>
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</div>
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<ul type="disc">
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  <ul type="circle">
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    <li>(Note: The orientation        of the BioBrick part should be opposite that of the auxotropic gene once        incorporated into the Sikorski vector.)</li>
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  </ul>
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</ul>
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<ul>
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  <li>Ligate the insert and vector  using standard techniques and transform into E. coli</li>
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  <li>Miniprep the vector from the  transformed strain</li>
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  <li>Linearize ~1 microgram of the  vector per transformation using the appropriate enzyme according to the  following table:</li>
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</ul>
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<p>&nbsp;</p>
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<table border="1" cellspacing="0" cellpadding="0">
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  <tr>
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    <td width="213" valign="top"><p>Vector</p></td>
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    <td width="213" valign="top"><p>Marker</p></td>
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    <td width="213" valign="top"><p>Linearization    Enzyme</p></td>
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  </tr>
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  <tr>
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    <td width="213" valign="top"><p>pRS303</p></td>
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    <td width="213" valign="top"><p>HIS3</p></td>
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    <td width="213" valign="top"><p>PstI</p></td>
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  </tr>
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  <tr>
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    <td width="213" valign="top"><p>pRS304*</p></td>
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    <td width="213" valign="top"><p>TRP1</p></td>
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    <td width="213" valign="top"><p>PstI</p></td>
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  </tr>
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  <tr>
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    <td width="213" valign="top"><p>pRS305</p></td>
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    <td width="213" valign="top"><p>LEU2</p></td>
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    <td width="213" valign="top"><p>BstEII</p></td>
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  </tr>
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  <tr>
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    <td width="213" valign="top"><p>pRS306</p></td>
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    <td width="213" valign="top"><p>URA3</p></td>
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    <td width="213" valign="top"><p>PstI</p></td>
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  </tr>
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</table>
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<ul>
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  <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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</ul>
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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>
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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 />
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  [2] <a href="http://openwetware.org/wiki/Silver:_BB_Strategy">http://openwetware.org/wiki/Silver:_BB_Strategy</a></p>
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</html>
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<p>The general layout of our limiter circuit is very amenable to variation.  Each of the logical roles can be filled by a number of different parts, possibly resulting in different behavior.  This process was made especially straightforward by the modular transcriptional control elements at our disposal.  Work with these transcription factors in the Silver Lab at Harvard showed that they show efficacy in regulating a variety of promoters, including the minimal mCYC1 and the inducible pGAL1.  To extend this work further, we have constructed a library of regulable promoters for use in our network, ligating binding sites for the synthetic transcription factors onto a variety of yeast promoters.</p>
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<p>mCYC was used as a minimal promoter, with all transcriptional induction and repression coming from transcription factors binding to sites ligated upstream.  We generated a variety of minimal promoters for use in the limiter system, with mCYC ligated to each of the four sets of binding sites, and three variations on mCYC ligated to two different sets of binding sites.  These will allow for construction of multiple variations on the limiter circuit.
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</p>
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<p>In a limiter circuit with a static threshold, the Thresh. promoter above is constitutive, but also requires regulation from alpha.  To investigate the effect of changing the threshold, we ligated binding sites onto two constitutive promoters: the high-level TEF2, and the low-level ADH1.</p>
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<p>Work in the Silver Lab has shown success in using binding sites to regulate inducible promoters.  We have constructed two versions of the MET25 promoter with binding sites, hoping to construct a limiter circuit in which we can set the threshold by varying the input concentration of methionine.

Revision as of 03:41, 30 October 2008



A Modular Approach to Constructing the Circuit

Limiter diagram.PNG


The general layout of our limiter circuit is very amenable to variation. Each of the logical roles can be filled by a number of different parts, possibly resulting in different behavior. This process was made especially straightforward by the modular transcriptional control elements at our disposal. Work with these transcription factors in the Silver Lab at Harvard showed that they show efficacy in regulating a variety of promoters, including the minimal mCYC1 and the inducible pGAL1. To extend this work further, we have constructed a library of regulable promoters for use in our network, ligating binding sites for the synthetic transcription factors onto a variety of yeast promoters.

mCYC was used as a minimal promoter, with all transcriptional induction and repression coming from transcription factors binding to sites ligated upstream. We generated a variety of minimal promoters for use in the limiter system, with mCYC ligated to each of the four sets of binding sites, and three variations on mCYC ligated to two different sets of binding sites. These will allow for construction of multiple variations on the limiter circuit.

In a limiter circuit with a static threshold, the Thresh. promoter above is constitutive, but also requires regulation from alpha. To investigate the effect of changing the threshold, we ligated binding sites onto two constitutive promoters: the high-level TEF2, and the low-level ADH1.

Work in the Silver Lab has shown success in using binding sites to regulate inducible promoters. We have constructed two versions of the MET25 promoter with binding sites, hoping to construct a limiter circuit in which we can set the threshold by varying the input concentration of methionine.
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