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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.

    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

    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 10, 2010 Digested PCR product and pSB1C3 with NotI, ligated and Transformed

    June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrA.Bfu_p1003+ and PrB'Bfu_p1003-

    June 10, 2010 PCRed p1003 (Kanamycin cassette)with primers PrB.Bfu_p1003+ and PrA'Bfu_p1003-

    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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