Team:WashU StLouis/Parts

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#groupparts {text-align: center; margin-left: auto; margin-right: auto;}
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<h1 >WELCOME TO iGEM 2014! </h1>
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<p>Your team has been approved and you are ready to start the iGEM season!
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<br>On this page you can document your project, introduce your team members, document your progress <br> and share your iGEM experience with the rest of the world! </p>
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<br>
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<p style="color:#E7E7E7"> <a href="https://2014.igem.org/wiki/index.php?title=Team:WashU_StLouis/Parts&action=edit"style="color:#FFFFFF"> Click here  to edit this page!</a> </p>
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<h1>Biobrick Cloning</h1>
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<a href="https://2014.igem.org/Team:WashU_StLouis"style="color:#000000">Home </a> </td>
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<a href="https://2014.igem.org/Team:WashU_StLouis/Team"style="color:#000000"> Team </a> </td>
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<a href="https://igem.org/Team.cgi?year=2014&team_name=WashU_StLouis"style="color:#000000"> Official Team Profile </a></td>
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<a href="https://2014.igem.org/Team:WashU_StLouis/Project"style="color:#000000"> Project</a></td>
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<a href="https://2014.igem.org/Team:WashU_StLouis/Parts"style="color:#000000"> Parts</a></td>
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<a href="https://2014.igem.org/Team:WashU_StLouis/Modeling"style="color:#000000"> Modeling</a></td>
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<a href="https://2014.igem.org/Team:WashU_StLouis/Notebook"style="color:#000000"> Notebook</a></td>
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<a href="https://2014.igem.org/Team:WashU_StLouis/Safety"style=" color:#000000"> Safety </a></td>
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<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>
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<a href="https://2014.igem.org/Team:WashU_StLouis/Attributions"style="color:#000000"> Attributions </a></td>
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<td align ="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td>
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<div style="text-align: center;"> <span
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style="font-weight: bold;">Light Regulation Group - Benjamin Huang</span><br>
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</div>
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<div style="text-align: justify;">Cloning the biobricks was more
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difficult than expected. Ben tried to do digestion/ligation using the
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restriction enzymes using the PSB1C3 backbone from <a href=http://parts.igem.org/Part:BBa_K1017726>BBa_K1017726</a>, but
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transformation into <span style="font-style: italic;">E. coli</span>
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strain DH10B yielded low efficiency with minimal number of colonies. He
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ran sequencing PCR with primers that bound to both the backbone and the
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target part. He found the prefix but not the suffix.<br>
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<br>
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Ben and Cheryl tried different sequencing primers that bound to sites
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on the genes instead, and re-ran sequencing PCR to no avail. Next, they
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tried religating using higher molar ratios 3:1 vector to insert, and
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the sequencing PCR ran into the same issue.<br>
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<br>
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We then got a new purified backbone from the distribution well from
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caroline and used that to clone. We also used Andrew's <a
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href="https://2014.igem.org/Team:WashU_StLouis/Protocol#17">CPEC</a>
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protocol to clone both the hybrid and normal part. We weren't able to
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clone the positive control because for the protocol you need 15-20 bp
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overlaps and the primers we had on hand did not allow for that. Also
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there was a very similar part on the parts registry so there was no use
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sending a part that essentially had the same Ptet and EYFP. <br>
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<br>
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Using PCR purified products, the CPEC cloning had much higher
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efficiency than digestion/ligation. We ran colony PCR on the colonies,
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checking for both the prefix and suffix, and the colonies that came
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from the CPEC had the right bands. We sent in the sequences for
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sequence verification and successfully sent in 2 biobrick parts.<br>
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</div>
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<br>
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<br>
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</td>
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<td style="width: 20%;"> <img style="width: 100%; height: 400px;"
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alt="Biobrick Submission"
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src="https://static.igem.org/mediawiki/2014/f/f9/WashU_biobrick.jpg"><br>
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<div style="text-align: center;">Biobrick Submission: 10/8/14<br>
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</div>
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</td>
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<td style="vertical-align: top; text-align: justify; width: 40%;">
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<div style="text-align: center; font-weight: bold;">Nitrogenase
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Group - Caroline Focht<br>
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</div>
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The original idea was to break the nif cluster from <i>Cyanothece </i>sp.
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51142 into six different parts since the entire cluster is upwards of
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35 kb. Primers were designed, and the desired fragments were produced
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via PCR. After running a gel to confirm the products, it was observed
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that some of the PCR reactions had not produced any of the desired
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fragments. <br>
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<br>
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Using a temperature gradient and DMSO, all of the fragments
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were eventually obtained. The BioBrick backbone pSC1B3 was isolated
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from an existing BioBrick part taken from the distribution plate using
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PCR and the codes for the BioBrick prefix and suffix as primers. This
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successfully produced the pSC1B3 plasmid, into which the six different
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fragments were inserted using Andrew Ng’s <a
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href="https://2014.igem.org/Team:WashU_StLouis/Protocol#17">CPEC</a>
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protocol. <br>
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<br>
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The now
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complete plasmids, backbone plus insert, were transformed into the <i>E.
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coli</i> strain XL1 Blue and plated on LB plates. The <i>E. coli</i> were
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cultured
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overnight, and four cultures from each plate were selected for colony
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PCR the next morning. The colony PCR indicated some positive results,
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and the plasmids were extracted from those strains, digested with the
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enzymes XbaI and SpeI, and confirmed with a gel. <br>
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<br>
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Only two of the
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BioBricks showed correct bands from the enzyme digest. After all of
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this was complete, it was discovered that all of our prospective
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BioBricks including the two successful ones, all contained one or more
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illegal restriction sites, thus invalidating their submission.</td>
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<td colspan="3" height="15"> Submitted Biobricks:<br>
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BBa_K1385000: CpcG2 promoter expressing TetR<br>
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BBa_K1385001: Hybrid CpcG2 promoter expressing TetR<br>
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<!--Parts Submitted to the Registry  -->
 
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<tr><td > <h3> Parts Submitted to the Registry </h3></td>
 
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<td ></td >
 
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<td > <h3>What information do I need to start putting my parts on the Registry? </h3></td>
 
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<p>
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<h3> Parts Table</h3>
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An important aspect of the iGEM competition is the use and creation of standard  biological parts. Each team will make new parts during iGEM and will submit them to the <a href="http://partsregistry.org"> Registry of Standard Biological Parts</a>. The iGEM software provides an easy way to present the parts your team has created. The "groupparts" tag will generate a table with all of the parts that your team adds to your team sandbox. 
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<p>
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<strong>Note that if you want to document a part you need to document it on the <a href="http://partsregistry.org Registry"> Registry</a>, not on your team wiki.</strong> Future teams and other users and are much more likely to find parts on the Registry than on your team wiki.
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</p>
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Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without a need to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.
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</p>
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<h3>When should you put parts into the Registry?</h3>
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<p>
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As soon as possible! We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better recall you will have of all details surrounding your parts. Remember you don't need to send us the DNA to create an entry for a part on the Registry. However, you must send us the sample/DNA before the Jamboree. Only parts for which you have sent us samples/DNA are eligible for awards and medal requirements.
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<td > </td>
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<p>
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The information needed to initially create a part on the Registry is:
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</p>
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<ol>
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<li>Part Name</li>
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<li>Part type</li>
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<li>Creator</li>
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<li>Sequence</li>
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<li>Short Description (60 characters on what the DNA does)</li>
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<li>Long Description (Longer description of what the DNA does)</li>
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<li>Design considerations</li>
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</ol>
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<p>
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. Check out part <a href="http://parts.igem.org/Part:BBa_K404003">BBa_K404003</a> for an excellent example of a highly characterized part.
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You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry"> Add a Part to the Registry</a> link.
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<td colspan="3" valign="top" width="45%">Any parts your team has
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created will appear in this table below:</td>
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<tr><td colspan="3" > <h3> Parts Table</h3></td></tr>
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<tr><td width="45%" colspan="3"  valign="top">
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Any parts your team has created will appear in this table below:</td></tr>
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<groupparts>iGEM014 WashU_StLouis</groupparts>
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<groupparts>iGEM013 WashU_StLouis</groupparts>
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<html> </table> </html>
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{{:Team:WashU_StLouis/footer}}

Latest revision as of 18:41, 16 October 2014



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