Team:Duke/Parts
From 2014.igem.org
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+ | <!--https://2014.igem.org/File:4oo8doudna.png--> | ||
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<h2> Construction Background </h2> | <h2> Construction Background </h2> | ||
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- | < | + | <p>During the summer we tested repression of GFP (pSB6A1-K608012) by pdCas9 with crRNA targeting the sequences below.</p> |
+ | <table> | ||
+ | <tr><td><span style="font-weight:bold">Sequence</span></td> <td><span style="font-weight:bold">Ungated Mean</span></td><td><span style="font-weight:bold">Fluorescence Relative to Reporter-Only</span></td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>Reporter</td><td>43.7</td><td>1</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>DH5alpha ZI (no GFP background)</td> <td>0.16</td><td>0.003661327231</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>dCas9</td><td>42.73333333</td><td>0.9778794813</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>GFP1 (ccatctaattcaacaagaat)</td><td>13.4</td><td>0.3066361556</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>GFP2 (agtagtgcaaataaatttaa)</td><td>48.6</td><td>1.112128146</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>GFP3 (gtagtgacaagtgttggcca)</td><td>15.43333333</td><td>0.3531655225</td></tr> | ||
+ | </table> | ||
+ | <!--<table> | ||
+ | <tr><td>Sequence</td> <td>Ungated Mean</td></tr> | ||
+ | <tr><td>DH5alpha ZI</td> <td>0.16</td></tr> | ||
+ | <tr><td>fluorescence relative to reporter-only</td> <td>0.003661327231</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>Reporter</td><td>43.7</td></tr> | ||
+ | <tr><td>fluorescence relative to reporter-only</td><td>1</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>dCas9</td><td>42.73333333</td></tr> | ||
+ | <tr><td>fluorescence relative to reporter-only</td><td>0.9778794813</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>GFP1 (ccatctaattcaacaagaat)</td><td>13.4</td></tr> | ||
+ | <tr><td>fluorescence relative to reporter-only</td><td>0.3066361556</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>GFP2 (agtagtgcaaataaatttaa)</td><td>48.6</td></tr> | ||
+ | <tr><td>fluorescence relative to reporter-only</td><td>1.112128146</td></tr> | ||
+ | <tr><td></td><td></td></tr> | ||
+ | <tr><td>GFP3 (gtagtgacaagtgttggcca)</td><td>15.43333333</td></tr> | ||
+ | <tr><td>fluorescence relative to reporter-only</td><td>0.3531655225</td></tr> | ||
+ | </table>--> | ||
+ | <p>We chose to first test decoy binding sites for GFP1 crRNA and designed an assembly scheme that proceeds as follows:</p> | ||
+ | |||
+ | <p>PCR1: oligos with prefix-decoy-spacer (top strand), decoy-spacer-decoy (top strand), and spacer-decoy-spacer (bottom strand) are combined and cycled 35 times resulting in a mixture of products</p> | ||
+ | <!--https://2014.igem.org/File:Parts1.png--> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/5/5b/Parts1.png" /> | ||
+ | <p>PCR2: the products of PCR1 are used as a template for a bottom strand oligo that appends the BioBrick suffix and cycles 35 times. </p> | ||
+ | <!--https://2014.igem.org/File:DukeParts2.png--> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/4/40/DukeParts2.png" /> | ||
+ | <p>The products of PCR2 are inserted in to pSB1C3 via Gibson assembly</p> | ||
+ | <!--https://2014.igem.org/File:Parts3.png--> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/3/34/Parts3.png" /> | ||
+ | <p>These constructs can be expanded further by serial digestion/ligation</p> | ||
+ | <!--https://2014.igem.org/File:Parts4.png--> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/cf/Parts4.png" /> | ||
+ | <p>From our PCR-based construction scheme we isolated 1x and 6x decoys, and then expanded the 6x array to 12x. These parts were submitted to the registry as BBa_K1545000 BBa_K1545001 and BBa_K1545001</p> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/3/3b/4oo8doudna.png" /> | ||
+ | <p>Crystal structure 4OO8 from the Doudna Lab. Cas9 is in blue, gRNA in red, and the DNA to which the complex binds is in yellow. <a href="http://www.ncbi.nlm.nih.gov/pubmed/24529477">Click for the PubMed article</a>. </p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <br /> | ||
+ | <h3>Parts Submitted to the Parts Registry </h3> | ||
<groupparts><a href="http://parts.igem.org/Part:BBa_K1545000">BBa_K1545000</a></groupparts> <br /> | <groupparts><a href="http://parts.igem.org/Part:BBa_K1545000">BBa_K1545000</a></groupparts> <br /> | ||
<groupparts><a href="http://parts.igem.org/Part:BBa_K1545001">BBa_K1545001</a></groupparts> <br /> | <groupparts><a href="http://parts.igem.org/Part:BBa_K1545001">BBa_K1545001</a></groupparts> <br /> |
Latest revision as of 03:53, 18 October 2014
Construction Background
During the summer we tested repression of GFP (pSB6A1-K608012) by pdCas9 with crRNA targeting the sequences below.
Sequence | Ungated Mean | Fluorescence Relative to Reporter-Only |
Reporter | 43.7 | 1 |
DH5alpha ZI (no GFP background) | 0.16 | 0.003661327231 |
dCas9 | 42.73333333 | 0.9778794813 |
GFP1 (ccatctaattcaacaagaat) | 13.4 | 0.3066361556 |
GFP2 (agtagtgcaaataaatttaa) | 48.6 | 1.112128146 |
GFP3 (gtagtgacaagtgttggcca) | 15.43333333 | 0.3531655225 |
We chose to first test decoy binding sites for GFP1 crRNA and designed an assembly scheme that proceeds as follows:
PCR1: oligos with prefix-decoy-spacer (top strand), decoy-spacer-decoy (top strand), and spacer-decoy-spacer (bottom strand) are combined and cycled 35 times resulting in a mixture of products
![](https://static.igem.org/mediawiki/2014/5/5b/Parts1.png)
PCR2: the products of PCR1 are used as a template for a bottom strand oligo that appends the BioBrick suffix and cycles 35 times.
![](https://static.igem.org/mediawiki/2014/4/40/DukeParts2.png)
The products of PCR2 are inserted in to pSB1C3 via Gibson assembly
![](https://static.igem.org/mediawiki/2014/3/34/Parts3.png)
These constructs can be expanded further by serial digestion/ligation
![](https://static.igem.org/mediawiki/2014/c/cf/Parts4.png)
From our PCR-based construction scheme we isolated 1x and 6x decoys, and then expanded the 6x array to 12x. These parts were submitted to the registry as BBa_K1545000 BBa_K1545001 and BBa_K1545001
![](https://static.igem.org/mediawiki/2014/3/3b/4oo8doudna.png)
Crystal structure 4OO8 from the Doudna Lab. Cas9 is in blue, gRNA in red, and the DNA to which the complex binds is in yellow. Click for the PubMed article.