Difference between revisions of "Team:MIT/Part Collection"

 
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<h1 style="color:#f20253; text-align: center; font-size: 40px; line-height: 40px;">Mammalian Synthetic Biology Parts <br> Starter Kit</h1>
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<h2><center><font size = "8"> Parts Collection - Mammalian Toolbox </font></center></h2>
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<p>With the development of efficacious gene editing tools and improved practices, mammalian synthetic biology grows more prevalent.  While our project is one example of how synthetic biology can be used to diagnose diseases, we hope that more people are able to build off our work, and the work of others to apply synthetic biology to mammalian cells.  With more work, we can come up with better, faster, less invasive diagnostics, and if these systems are refined, we could potentially apply the same genetic circuit techniques to the field of treatments.</p>
  
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<p>Hoping to see what amazing work can come from future teams and scientists, we have put together a <a href="https://2016.igem.org/Team:MIT/Part_Collection">toolbox of parts</a> to make it easier to start mammalian work.  </p>  
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<table>
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<p>Our kit is meant to be used with <a href="https://2008.igem.org/Team:UC_Berkeley/GatewayOverview">Gateway cloning reactions</a>.  Gateway is a recombinase-based cloning method used by the phage λ against e.Coli, and isolated and optimized for for synthetic biology by Invitrogen.
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</p>
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<th><font size = "4">
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Part Name </font>
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Part Number</font>
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<td>pENTR hEF1a</td>
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100003"> BBa_K2100003</a></td>
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<td> pENTR pTRE</td>
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100004">BBa_K2100004</a></td>
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<p>
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<div class = "column half_size">
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<img src="https://static.igem.org/mediawiki/2016/0/09/T--MIT--pEXPR2016.svg">
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</div>
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<div class = "column half_size">
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<p>There are two plasmids involved:
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<li>pENTR: the entry plasmids contain the desired genes, flanked by L1 and L2 sites.</li>
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<li>pDEST: the destination plasmid will eventually be transformed into the E.Coli.  There is a selection or screening tool flanked by R2 and R4 sites.</li>
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<p>From recombination of the L4, R1, L1, L2, R4 and R2 sites, the pEXPR expression plasmid is formed. </p> 
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<img src="https://static.igem.org/mediawiki/2016/5/55/T--MIT--pEXPRproduct2016.png" style="width:375px;height:390px;">
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</div>
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</p>
  
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<p> </p>
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<td> pENTR pERE3</td>
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100000">BBa_K2100000</a></td>
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<p> Currently, the selection tool used for the pDEST is ccdB.  However, a <a href="http://tools.thermofisher.com/content/sfs/manuals/11808011.pdf">patent</a> on it, giving Invitrogen the proprietor of ccdB as a selection tool makes it impossible to distribute to iGEM teams.  Because of this, iGEM had to discontinue <a href="http://parts.igem.org/Part:BBa_P1010">multiple parts </a>containing ccdB in their registry, and to avoid legal issues: </p>  
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<td> pENTR pERE5</td>
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100001">BBa_K2100001</a></td>
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<img src = "https://static.igem.org/mediawiki/2016/9/9f/T--MIT--ccdBdiscontinued.png">
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<td> pENTR pERE6</td>
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100002">BBa_K2100002</a></td>
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<p>To allow distribution, we have created a <a href="http://parts.igem.org/Part:BBa_K2100015">pDEST plasmid</a> that has mCherry fluorescence as a screening tool. </p>
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<td> pENTR pPRE3</td>
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100008">BBa_K2100008</a></td>
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<td> pENTR pPRE4</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100009">BBa_K2100009</a></td>
 
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<center><img src="https://static.igem.org/mediawiki/2016/a/a2/T--MIT--pDestmCherry2016.svg" style="width:653px;height:518px;"></center></a>
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<td> pENTR pHybrid</td>
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100010">BBa_K2100010</a></td>
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<p>Along with our pDEST, we include several pENTR vectors, with promoters and useful genes that can be combined and put into eColi to grow up before transfection into mammalian systems. <b>Click on each part to see the entry in iGEM's registry</b> </p>
  
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<td> pENTR pEGSH </td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100021">BBa_K2100021</a></td>
 
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<img src="https://static.igem.org/mediawiki/2016/c/c3/T--MIT--toolkitpartsformap.png" usemap="#image-map">
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<td> pENTR pBM3R1 </td>
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<map name="image-map">
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100022">BBa_K2100022</a></td>
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    <area target="_blank" alt="hEF1a constitutive promotor" title="hEF1a constitutive promotor" href="http://parts.igem.org/Part:BBa_K2100003" coords="287,182,23,101" shape="rect">
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    <area target="_blank" alt="mKate red fluorescence" title="mKate red fluorescence" href="http://parts.igem.org/Part:BBa_K2100006" coords="514,96,778,131" shape="rect">
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    <area target="_blank" alt="pBM3R1 repressible promoter" title="pBM3R1 repressible promoter" href="http://parts.igem.org/Part:BBa_K2100022" coords="26,384,435,470" shape="rect">
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    <area target="_blank" alt="pTALER14 repressible promoter" title="pTALER14 repressible promoter" href="http://parts.igem.org/Part:BBa_K2100024" coords="26,485,353,565" shape="rect">
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    <area target="_blank" alt="pTALER21 repressible promoter" title="pTALER21 repressible promoter" href="http://parts.igem.org/Part:BBa_K2100023" coords="25,574,360,661" shape="rect">
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    <area target="_blank" alt="pEGSH inducible promoter" title="pEGSH inducible promoter" href="http://parts.igem.org/Part:BBa_K2100021" coords="21,727,429,815" shape="rect">
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    <area target="_blank" alt="pTRE inducible promoter" title="pTRE inducible promoter" href="http://parts.igem.org/Part:BBa_K2100004" coords="22,831,438,941" shape="rect">
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    <area target="_blank" alt="pERE 3 repeat inducible promoter" title="pERE 3 repeat inducible promoter" href="http://parts.igem.org/Part:BBa_K2100000" coords="21,947,435,1043" shape="rect">
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    <area target="_blank" alt="pPRE 3 repeat inducible promoter" title="pPRE 3 repeat inducible promoter" href="http://parts.igem.org/Part:BBa_K2100008" coords="23,1054,434,1134" shape="rect">
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    <area target="_blank" alt="tagBFP fluorescence" title="tagBFP fluorescence" href="http://parts.igem.org/Part:BBa_K2100007" coords="516,133,777,151" shape="rect">
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    <area target="_blank" alt="eYFP fluorescence" title="eYFP fluorescence" href="http://parts.igem.org/Part:BBa_K2100005" coords="518,157,778,190" shape="rect">
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    <area target="_blank" alt="mCherry fluorescence" title="mCherry fluorescence" href="http://parts.igem.org/Part:BBa_K2100014" coords="518,191,775,225" shape="rect">
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    <area target="_blank" alt="GAL4-VP16 transactivator" title="GAL4-VP16 transactivator" href="http://parts.igem.org/Part:BBa_K2100020" coords="491,273,771,345" shape="rect">
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    <area target="_blank" alt="BM3R1 inducer" title="BM3R1 inducer" href="http://parts.igem.org/Part:BBa_K2100018" coords="493,347,769,416" shape="rect">
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    <area target="_blank" alt="TALER14 inducer" title="TALER14 inducer" href="http://parts.igem.org/Part:BBa_K2100016" coords="498,449,769,511" shape="rect">
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    <area target="_blank" alt="TALER21 inducer" title="TALER21 inducer" href="http://parts.igem.org/Part:BBa_K2100017" coords="500,544,767,612" shape="rect">
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    <area target="_blank" alt="VgEcR-RXR inducer" title="VgEcR-RXR inducer" href="http://parts.igem.org/Part:BBa_K2100026" coords="783,778,503,684" shape="rect">
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    <area target="_blank" alt="rtTA inducer" title="rtTA inducer" href="http://parts.igem.org/Part:BBa_K2100022" coords="499,811,783,880" shape="rect">
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    <area target="_blank" alt="TP901 recombinase" title="TP901 recombinase" href="http://parts.igem.org/Part:BBa_K2100019" coords="508,957,771,1023" shape="rect">
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</map>
  
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<p>Through an LR reaction, the pDEST mCherry combines with the pENTR gene and promoter plasmids.  In the process, the mCherry is cut out.  When eColi are transformed with the resulting plasmids and plated, the ones containing desired plasmids are white, while unsuccessful colonies fluoresce</p><center><img src ="https://static.igem.org/mediawiki/2016/f/f9/T--MIT--mCherryatlast.png"></center>
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<td> pENTR pTALER21</td>
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100023">BBa_K2100023</a></td>
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<td> pENTR pTALER14</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100024">BBa_K2100024</a></td>
 
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<td> pENTR eYFP</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100005">BBa_K2100005</a></td>
 
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</tr>
 
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<td> pENTR mKate</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100006">BBa_K2100006</a></td>
 
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</tr>
 
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<td> pENTR tagBFP</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100007">BBa_K2100007</a></td>
 
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</tr>
 
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<tr>
 
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<td> pENTR mCherry</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100014">BBa_K2100014</a></td>
 
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</tr>
 
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<td> pENTR TALER14</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100016">BBa_K2100016</a></td>
 
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</tr>
 
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<tr>
 
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<td> pENTR TALER21</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100017">BBa_K2100017</a></td>
 
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</tr>
 
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<td> pENTR BM3R1</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100018">BBa_K2100018</a></td>
 
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</tr>
 
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<tr>
 
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<td> pENTR TP901</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100019">BBa_K2100019</a></td>
 
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</tr>
 
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<tr>
 
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<td> pENTR GAL4 - VP16</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100020">BBa_K2100020</a></td>
 
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</tr>
 
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<td> pENTR rtTA</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100022">BBa_K2100022</a></td>
 
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</tr>
 
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<td> pEXPR hEF1a-eYFP</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100011">BBa_K2100011</a></td>
 
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</tr>
 
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<tr>
 
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<td> pEXPR hEF1a-mKate</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100012">BBa_K2100012</a></td>
 
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</tr>
 
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<td> pEXPR hEF1a-tagBFP</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100013">BBa_K2100013</a></td>
 
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</tr>
 
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<td> pDEST mCherry GG</td>
 
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<td> <a href = "http://parts.igem.org/Part:BBa_K2100015">BBa_K2100015</a></td>
 
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<p>Did your team make a lot of great parts? Is there a theme that ties all your parts together? Do you have more than 10 parts in this collection? Did you make a CRISPR collection, a MoClo collection, or a collection of awesome pigment parts? Describe your parts collection on this page, so the judges can evaluate you for the Best Part Collection award.</p>
 
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While you should put all the characterization information for your parts on the Registry, you are encouraged to explain how all your parts form a collection on this page.
 
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Latest revision as of 03:09, 20 October 2016

Mammalian Synthetic Biology Parts
Starter Kit

With the development of efficacious gene editing tools and improved practices, mammalian synthetic biology grows more prevalent. While our project is one example of how synthetic biology can be used to diagnose diseases, we hope that more people are able to build off our work, and the work of others to apply synthetic biology to mammalian cells. With more work, we can come up with better, faster, less invasive diagnostics, and if these systems are refined, we could potentially apply the same genetic circuit techniques to the field of treatments.

Hoping to see what amazing work can come from future teams and scientists, we have put together a toolbox of parts to make it easier to start mammalian work.

Our kit is meant to be used with Gateway cloning reactions. Gateway is a recombinase-based cloning method used by the phage λ against e.Coli, and isolated and optimized for for synthetic biology by Invitrogen.

There are two plasmids involved:

  • pENTR: the entry plasmids contain the desired genes, flanked by L1 and L2 sites.
  • pDEST: the destination plasmid will eventually be transformed into the E.Coli. There is a selection or screening tool flanked by R2 and R4 sites.

    • From recombination of the L4, R1, L1, L2, R4 and R2 sites, the pEXPR expression plasmid is formed.

    Currently, the selection tool used for the pDEST is ccdB. However, a patent on it, giving Invitrogen the proprietor of ccdB as a selection tool makes it impossible to distribute to iGEM teams. Because of this, iGEM had to discontinue multiple parts containing ccdB in their registry, and to avoid legal issues:

    To allow distribution, we have created a pDEST plasmid that has mCherry fluorescence as a screening tool.

    Along with our pDEST, we include several pENTR vectors, with promoters and useful genes that can be combined and put into eColi to grow up before transfection into mammalian systems. Click on each part to see the entry in iGEM's registry

    hEF1a constitutive promotor mKate red fluorescence pBM3R1 repressible promoter pTALER14 repressible promoter pTALER21 repressible promoter pEGSH inducible promoter pTRE inducible promoter pERE 3 repeat inducible promoter pPRE 3 repeat inducible promoter tagBFP fluorescence eYFP fluorescence mCherry fluorescence GAL4-VP16 transactivator BM3R1 inducer TALER14 inducer TALER21 inducer VgEcR-RXR inducer rtTA inducer TP901 recombinase

    Through an LR reaction, the pDEST mCherry combines with the pENTR gene and promoter plasmids. In the process, the mCherry is cut out. When eColi are transformed with the resulting plasmids and plated, the ones containing desired plasmids are white, while unsuccessful colonies fluoresce