Team:UC Davis/notebook/assembly.html
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<a name="assembly"></a><p class="header"><b>Assembly Workplans</b></a><p> | <a name="assembly"></a><p class="header"><b>Assembly Workplans</b></a><p> | ||
+ | |||
+ | <a name="pieces"></a><p class="header"><b>Creating Each Individual Composite Part</b></a><p> | ||
+ | |||
+ | <b>**Click on the individual pieces below to view how we ligated them together.**</b> <p><br /> | ||
+ | <b>Miscellaneous Parts</b></a><br /> | ||
+ | <table border=0 margin="0" width="420px" padding:0 margin:0> | ||
+ | <tr> | ||
+ | <td><a href="https://2010.igem.org/Team:UC_Davis/notebook/lightsensor2.html" class="help"><img src="https://static.igem.org/mediawiki/2010/2/29/Lightsensorbutton.jpg"></a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td width="200px"><a href="https://2010.igem.org/Team:UC_Davis/notebook/luxreceiver.html" class="help"><img src="https://static.igem.org/mediawiki/2010/4/43/Luxreceiverbutton.jpg"></a></td> | ||
+ | <td width="200px"><a href="https://2010.igem.org/Team:UC_Davis/notebook/lasreceiver.html" class="help"><img src="https://static.igem.org/mediawiki/2010/5/5e/Lasreceiverbutton.jpg"></a></td> | ||
+ | </tr></table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table><p><br /> | ||
+ | |||
+ | <b>Genetic Circuit Pieces</b><br /> | ||
+ | <table border=0 margin="0" width="405px" padding:0 margin:0> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td width="200px"><a href="https://2010.igem.org/Team:UC_Davis/notebook/initiatorx.html" class="help"><img src="https://static.igem.org/mediawiki/2010/0/0a/Initiatorbutton.jpg"></a></td> | ||
+ | <td width="200px"><a href="https://2010.igem.org/Team:UC_Davis/notebook/lassensor.html" class="help"><img src="https://static.igem.org/mediawiki/2010/7/72/Lassensorbutton.jpg"></a></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://2010.igem.org/Team:UC_Davis/notebook/onpathway.html" class="help"><img src="https://static.igem.org/mediawiki/2010/d/d8/Onswitchbutton.jpg"></a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://2010.igem.org/Team:UC_Davis/notebook/luxpathway.html" class="help"><img src="https://static.igem.org/mediawiki/2010/2/23/Offswitchbutton.jpg"></a></td> | ||
+ | </tr> | ||
+ | </table><p><br /> | ||
+ | |||
+ | |||
+ | <a name="plasmid"></a><p class="header"><b>Next Mission: Placing them into only two plasmids</b></a><p> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/3/36/Plasmid1.jpg"><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2010/1/12/Plasmid2.jpg"><p><br /> | ||
+ | |||
+ | |||
+ | <a name="topagar"></a><p class="header"><b>Final Mission: Create an Even Lawn of E. Coli</b><br /> | ||
<ul> | <ul> | ||
- | <li><a href="https://2010.igem.org/Team:UC_Davis/ | + | <li>We are using this <a href="https://2010.igem.org/Team:UC_Davis/protocols/topagar.html" class="help">top agar plating technique </a></li> |
- | <li><a href="https://2010.igem.org/Team:UC_Davis/ | + | <li>Next is to test the what the optimal OD, arabinose concentration, and amount of light it takes to a) create an even lawn of E. Coli and b) see contrast between the "on" and "off" cells </li> |
- | <li><a href="https://2010.igem.org/Team:UC_Davis/ | + | </ul><p><br /> |
+ | |||
+ | |||
+ | <a name="protocols"></a><p class="header"><b>Basic Assembly Protocols</b></a><p> | ||
+ | <b>For more insight about the process behind the ligations, here is a sample ligation procedure. Because the initiator is one of the smaller constructs, we have decided to use it as an example to illustrate the protocols used in lab.</b><p> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/2/29/Initiatorconstruct.jpg"><p> | ||
+ | |||
+ | <a name="startup"><h1>Starting Up</h1></a> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/hydration.html" class="help">Hydrated</a> parts: BBa_B0034, BBa_C0051, BBa_B0015, BBa_R0082.</li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">Transformed</a> these parts into DH5α competent cells.</li> | ||
+ | </ul><p><br /> | ||
+ | |||
+ | <a name="rbsc0051"><h1>RBS to BBa_C0051</h1></a> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: BBa_B0034 and BBa_C0051</li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> BBa_B0034 and BBa_C0051</li> | ||
</ul> | </ul> | ||
+ | |||
+ | <table border="0" margin="1" width="650px" padding="5px"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/f/f6/RBSplasmidclosedxx.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with SpeI & Pst</li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/2/29/RBSplasmidx.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help">Run on gel</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/2/29/RBSplasmidx.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/d/d3/C0051plasmidclosed.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with Xba & Pst</li></ul> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/3/3d/C0051plasmidcut.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li>Separate fragments through <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help"> gel electrophoresis</a></li><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/c/c5/C0051insert1.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table><p><br /> | ||
+ | |||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the RBS and BBa_C0051 fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on carb plates.</li> | ||
+ | <li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed. | ||
+ | </ul><br /> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/b/bb/RBSc0051.jpg"><br /> | ||
+ | |||
+ | Assembly notes | ||
+ | <ul> | ||
+ | <li>It is most practical to cut RBS as a vector because RBS is less than 100 bp long as an insert, making it hard to see and extract on the gel.</li> | ||
+ | <li>The sticky ends from the XbaI and SpeI, when ligated together, will form an 8 bp scar. Thus, XbaI and SpeI will no longer be able to cut here.</li> | ||
+ | </ul><br /> | ||
+ | |||
+ | <a name="stop"><h1>'RBS & BBa_C0051' to Terminator</h1></a> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: 'RBS & BBa_C0051', BBa_B0015</li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> 'RBS & BBa_C0051' and BBa_B0015</li> | ||
+ | </ul> | ||
+ | |||
+ | <table border="0" margin="1" width="650px" padding="5px"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/9/9a/Stopplasmid.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with EcoRI & XbaI</li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/7/70/Xstopopenvector.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help">Run on gel</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/7/70/Xstopopenvector.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/b/bb/RBSc0051.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with EcoRI & SpeI</li></ul> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/9/94/RBSc0051cut.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li>Separate fragments through <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help"> gel electrophoresis</a></li><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/d/de/RBSc0051insertx.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table><p><br /> | ||
+ | |||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on kan plates.</li> | ||
+ | <li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed. | ||
+ | </ul><br /> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/e/eb/RBSc0051stop.jpg"><br /><p> | ||
+ | |||
+ | Assembly notes <br /> | ||
+ | <ul> | ||
+ | <li>It is most practical to cut BBa_B0015 as the vector because it is only 130 bp long. It would be hard to see on a gel. </li> | ||
+ | </ul><br /> | ||
+ | |||
+ | <a name="r0082"><h1>'RBS & BBa_C0051 & stop' to BBa_R0082</h1></a> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: 'RBS & BBa_C0051 & stop', BBa_R0082</li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> 'RBS & BBa_C0051& stop' and BBa_R0082</li> | ||
+ | </ul> | ||
+ | |||
+ | <table border="0" margin="1" width="650px" padding="5px"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/c/cc/Promoterclosed.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with SpeI & PstI</li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/2/21/Promoterplasmidopenx.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help">Run on gel</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/2/21/Promoterplasmidopenx.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/e/eb/RBSc0051stop.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with XbaI & PstI</li></ul> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/4/46/RBSc0051stopcut.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li>Separate fragments through <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help"> gel electrophoresis</a></li><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/6/61/RBSc0051stopinsertx.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table><p><br /> | ||
+ | |||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on carb plates.</li> | ||
+ | <li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed. | ||
+ | </ul><br /> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/3/34/Initiatorplasmid.jpg"><br /> | ||
+ | |||
+ | Assembly notes | ||
+ | <ul> | ||
+ | <li>The promoter had to be cut as a vector because it is only 108 bp by itself.</li> | ||
+ | </ul><br /> | ||
<p> | <p> | ||
Line 45: | Line 294: | ||
<td class="kirby"> | <td class="kirby"> | ||
<ul> | <ul> | ||
- | <li>Assembly Workplan</li> | + | <li><a href="#assembly" class="help">Assembly Workplan</li> |
+ | <li><a href="#pieces" class="help">Creating Each Composite Part</li> | ||
+ | <li><a href="#plasmid" class="help">Placing Into Two Plasmids</li> | ||
+ | <li><a href="#topagar" class="help">Creating Lawn of E. Coli</li> | ||
+ | <li><a href="#protocols" class="help">Basic Assembly Protocols</li> | ||
</ul> </td> | </ul> </td> | ||
</tr> | </tr> |
Latest revision as of 22:19, 27 October 2010
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