Team:Alberta/Notebook

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<p class="logo"><img src="https://static.igem.org/mediawiki/2010/c/cc/Alberta_Logo.png" width="50px" height="50px" padding="5px" align="left"></img></p>
 
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genomikon</div>
 
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<div id="navbar"><span class="llink2"><a href="https://2010.igem.org/Team:Alberta/JTest"> home</a> |  <a href="project"> project</a> | <a href="ethics"> ethics</a> | <a href="parts"> parts</a> | <a href="software"> software</a> | <a href="https://2010.igem.org/Team:Alberta/Notebook"> notebook</a> | <a href="team"> team</a></span>
 
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  <li onclick="showTab('vtab1')">iGEM 2010 Notebook</li>
 
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  <li onclick="showTab('vtab2')">Building Parts</li>
 
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  <li onclick="showTab('vtab3')">Testing Parts</li>
 
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  <li onclick="showTab('vtab4')">Assembly Method</li>
 
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  <li onclick="showTab('vtab5')">Plates</li>
 
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  <li onclick="showTab('vtab6')">Competent Cells</li>
 
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<div id="vtab1" class="vtInfo"><span class="h2">iGEM 2010 Notebook</span>
 
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<br>
 
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<p>The lab notebook chronicles our journey in the creation of the Genomikon kit.  Many paths were woven together in space and time to reach this finished masterpiece.  To help you navigate through these trials with us we have laid out our notebook in a layered fashion.  Each This page gives a sketch of each project and how it interacts with each other.  Then follow the links to a projects page for time line of the major landmarks and accomplishments.  If you require more details on the project the links within that page will take you to our day-by-day work log.</p>
 
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<div id="vtab2" class="vtInfo"><span class="h2">Building Parts</span>
 
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<p>The Building Parts project was responsible for building the scaffold that is used to systematically produce both A and B type parts. The first version scaffold plasmids (pC.b.A-001 and pC.b.B-001)contained our own specialized prefix and suffix nested inside of the standard BioBrick prefix and suffix.  The second version scaffold plasmids (pC.b.A-006 and pC.b.B-006) contained a RFP coding device (BBa_J04550) between our prefix and suffix instead of kanamycin resistance.  These second generation parts plasmids were fantastic base plasmids from which we are able to amplify any part at all because it provided a selection marker when transformed into DH5&alpha; (ie. the colonies that are white not red contained a plasmid with a part). At this point we were able to make parts en masse to put in our kit. After obtaining a particular part in a plasmid we PCRed the part and digested, ready to use in Assembly or to Test the plasmid.</p>
 
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<img src="https://static.igem.org/mediawiki/2010/4/40/Alberta2010kaBuilding_parts_image.jpg" padding="0px" width="600px"></img>
 
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<div id="highlightb"></div>
 
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<span class="h3">Base Plasmids v.1</span>
 
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<p>Our original base plasmids contain a Kanamycin resistance cassette (p1003) bracketed by BsaI or BfuAI or BbsI cut sites.  If cut with either BsaI or BfuAI or BbsI, the Kanamycin cassette is release with sticky ends characteristic of an A or a B BioByte. </p>
 
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<b>BsaI plasmids</b>
 
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<p><b>May 10, 2010</b> PCRed p1003 (Kanamycin cassette)with primers PrA_p1003+ and PrB'_p1003-</p>
 
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<p><b>May 10, 2010</b> PCRed p1003 (Kanamycin cassette)with primers PrB_p1003+ and PrA'_p1003-</p>
 
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<p><b>May 27, 2010</b> Digested pSB1C3 and our PCR products of p1003 (Kanamycin cassette)with Not1 </p>
 
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<p><b>May 27, 2010</b> Ligated PCR products of p1003 (Kanamycin cassette) and pSB1C3 </p>
 
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<p><b>May 27, 2010</b> Transformed Ligation.
 
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<p><b>May 28, 2010</b> Success! We got colonies that grew on Kan/Chlor plates
 
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<p><b>May 30, 2010-June 9,2010</b> Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation of Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact</p>
 
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</br>
 
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<b>BbsI Plasmids</b>
 
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<p><b>June 10, 2010</b> PCRed p1003 (Kanamycin cassette)with primers PrA.Bbs_p1003+ and PrB'Bbs_p1003-</p>
 
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<p><b>June 10, 2010</b> PCRed p1003 (Kanamycin cassette)with primers PrB.Bbs_p1003+ and PrA'Bbs_p1003-</p>
 
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<p><b>June 14, 2010</b> Digested PCR product and pSB1C3 with NotI, ligated
 
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<p><b>June 16, 2010</b> Transformed from ligation
 
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<p><b>June 17, 2010</b> Success! We did manage to get colonies that grew on Kan/Chlor plates
 
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<p><b>June 18-21, 2010</b> Miniprep of colonies, Digested with EcoRI and XbaI to determine orientation of Kanamycin Cassette until found plasmids in which the BioBrick prefix and suffix remained intact</p>
 
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</br>
 
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<b>BfuAI Plasmids</b>
 
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<p><b>June ?, 2010</b> PCRed p1003 (Kanamycin cassette)with primers PrA.Bfu_p1003+ and PrB'Bfu_p1003-</p>
 
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<p><b>June ?, 2010</b> PCRed p1003 (Kanamycin cassette)with primers PrB.Bfu_p1003+ and PrA'Bfu_p1003-</p>
 
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</br>
 
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<p><b>Bsa AB chlor and tet parts</b></p>
 
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<p>The strategy is to digest Bsa AB kan and pSB1A3 with NotI, ligate, and then cut out the kan using BsaI and insert chlor or tet Bsa AB parts.
 
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<p><b>July 4, 2010</b>  Digested pre-PCR'ed kan Bsa AB fragment and pSB1A3 with NotI. Ligated kan and pSB1A3 together.
 
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<p><b>July 5, 2010</b>  Transformed from the ligation of kan and pSB1A3.
 
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<p><b>July 6, 2010</b>  Success! Numerous white colonies on all plates. Set up overnights of pSB1A3 with kan insert.
 
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<p><b>July 7, 2010</b>  Miniprepped overnights and digested with XbaI and PstI to check for orientation. Picked two tubes that were digested correctly and digested them with BsaI to remove kan. Ligated chlor Bsa AB and tet Bsa AB from previous PCR's with the PSB1A3 plasmid now lacking kan.
 
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<p><b>July 8, 2010</b>  Transformed from ligations of chlor and tet with pSB1A3 backbone.
 
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<p><b>July 9, 2010</b>  Success! Growth on all plates. Overnights were set up.
 
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<p><b>July 10, 2010</b> Minipreps were done and were digested with EcoRI to test for size. Three tubes of chlor Bsa AB and three of tet Bsa AB looked good.
 
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<p><b>July 20, 2010</b>  Glycerol stocks were made of chlor and tet Bsa AB.
 
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<p><b>August 9, 2010</b>  Sequenced Bsa AB amp, tet, and chlor. Success!
 
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<p><b>Bsa BA amp and tet</b>
 
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<div id="highlightb"></div>
 
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<span class="h3">Abandoned Projects</span>
 
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</br>
 
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<p><b>ccdB base plasmids</b></p>
 
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<p>The idea with this plasmid is that if we have a plasmid backbone that has ccdB in the place where A or B BioBytes would go, when we produce BioBytes, we will have a positive selection for plasmids with our BIobytes.
 
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<p><b>June 22, 2010</b> PCRed p1016 (ccdB cassette)with primers PrA.I52002+ and PrB.I52002-</p>
 
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<p><b>June 22, 2010</b> PCRed p1016 (ccdB cassette)with primers PrB.I52002+ and PrA.I52002-</p>
 
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<p><b>June 23, 2010</b> Digeseted PCR product with BsaI.  Digested Bsa base plasmid v.1 and Bbs base plasmid v.1 with Bsa and Bbs respectively.
 
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<p><b>June 24,2010</b> Ligated ccdB PCRs and base plasmids together. Transformed from the ligation into DBL3 cells. 
 
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<p><b>June 29-July 5,2010</b> Checked ccdB plasmids by streaking on both a Chlor plate and a Kan/Chlor plate.  Colonies that only grew on Chlor, miniprepped and digested with EcoRI and PstI to check for the insert
 
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<p>This project was abandoned because even if it appeared that the plasmids cut, we could not find a band for the ccdB gene.  We theorized that the ccdB gene was just unstable and didn't behave as expected... This may also have been the reason it was not put into the 2010 registry and we had to get it from the 2009 registry.  Live and learn</p>
 
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</br>
 
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<p><b>BFP plasmids</b></p>
 
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<p>When the ccd B plasmids were a flop, we decided that we would make our base plasmids with a Flourescent protein between the prefix and suffix.  If we used such a plasmid to put Biobytes into, we would have a selection measure to determine which plasmids contained our parts.
 
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<div id="vtab3" class="vtInfo"><span class="h2">Testing Parts</span> 
 
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<p>Before we were able to test parts we created 2 base testing plasmids (vector 01 and vector 02).  Vector 01 is designed to test Open Reading Frame parts, or parts that code for proteins.  The part is flanked by a promoter and the start codon on one side and a stop codon and terminator on the other.  Vector 02 is designed to test linker parts, or parts that control the expression of the Open Reading Frame parts they are next two.  In Vector 02 the part is flanked by two distinct reporter genes, that by comparing the relative expression of the 2 reporter genes we can determine the behavior of the linking part.</p>
 
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<img src="https://static.igem.org/mediawiki/2010/b/b1/Alberta_Oscar.png" width="400px" height="533px" padding="0px" align="left"></img>
 
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<span class="h2">Oscar Cortes</span><br>
 
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<span class="h3">Specialized in Molecular Genetics (Graduate)</span>
 
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<a href="Site_Map">site map</a>|<a href="Sponsors">sponsors</a>|<a href="Contact">contact us</a>
 
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[[page78]] [[Team:Alberta/Notebook_May|page79]]
 

Revision as of 22:02, 31 August 2010

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