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  • iGEM 2010 Notebook
  • Building Parts
  • Testing Parts
  • Assembly Method
  • Plates
  • Competent Cells
  • Software
  • iGEM 2010 Notebook

    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.

    Building Parts

    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 DH5α (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.

    Base Plasmids v.1

    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.

    BsaI plasmids

    May 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA_p1003+ and PrB'_p1003-

    May 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB_p1003+ and PrA'_p1003-

    May 27, 2010 Digested pSB1C3 and our PCR products of p1003 (Kanamycin cassette)with Not1

    May 27, 2010 Ligated PCR products of p1003 (Kanamycin cassette) and pSB1C3

    May 27, 2010 Transformed Ligation.

    May 28, 2010 Success! We got colonies that grew on Kan/Chlor plates

    May 30, 2010-June 9,2010 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

    BbsI Plasmids

    June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bbs_p1003+ and PrB'Bbs_p1003-

    June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bbs_p1003+ and PrA'Bbs_p1003-

    June 14, 2010 Digested PCR product and pSB1C3 with NotI, ligated

    June 16, 2010 Transformed from ligation

    June 17, 2010 Success! We did manage to get colonies that grew on Kan/Chlor plates

    June 18-21, 2010 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

    BfuAI Plasmids

    June ?, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bfu_p1003+ and PrB'Bfu_p1003-

    June ?, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bfu_p1003+ and PrA'Bfu_p1003-

    Bsa AB chlor and tet parts

    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.

    July 4, 2010 Digested pre-PCR'ed kan Bsa AB fragment and pSB1A3 with NotI. Ligated kan and pSB1A3 together.

    July 5, 2010 Transformed from the ligation of kan and pSB1A3.

    July 6, 2010 Success! Numerous white colonies on all plates. Set up overnights of pSB1A3 with kan insert.

    July 7, 2010 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.

    July 8, 2010 Transformed from ligations of chlor and tet with pSB1A3 backbone.

    July 9, 2010 Success! Growth on all plates. Overnights were set up.

    July 10, 2010 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.

    July 20, 2010 Glycerol stocks were made of chlor and tet Bsa AB.

    August 9, 2010 Sequenced Bsa AB amp, tet, and chlor. Success!

    Bsa BA amp and tet

    Abandoned Projects

    ccdB base plasmids

    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.

    June 22, 2010 PCRed p1016 (ccdB cassette)with primers PrA.I52002+ and PrB.I52002-

    June 22, 2010 PCRed p1016 (ccdB cassette)with primers PrB.I52002+ and PrA.I52002-

    June 23, 2010 Digeseted PCR product with BsaI. Digested Bsa base plasmid v.1 and Bbs base plasmid v.1 with Bsa and Bbs respectively.

    June 24,2010 Ligated ccdB PCRs and base plasmids together. Transformed from the ligation into DBL3 cells.

    June 29-July 5,2010 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

    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

    BFP plasmids

    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.

    Testing Parts

    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.

    Oscar Cortes
    Specialized in Molecular Genetics (Graduate)

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