Team:Alberta/Notebook August



August 2010

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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. 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 to first build a plasmid (plasmid 01)that contained our own specialized prefix and suffix nested inside of the standard BioBrick prefix and suffix. After plasmid 01 existed we inserted the CcdB gene (the "death" gene) between our prefix and suffix removing the gene for Kanamycin resistance (plasmid 02). Plasmid 02 is fantastic base plasmid from which we are able to amplify any part at all because it provides a positive selection marker when transformed into DH5α. 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 it ready to use in Assembly or to Test the plasmid.

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.

Assembly Method

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Competent Cells

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July 1, 2010

Building Parts

Made 5mL liquid cultures with Choramphenicol with streaks from plates of ccdB Bbs A/B', ccdB Bbs B/A', cddB BsaA/B', ccdB BsaB/A' part plasmids from 25-06-2010. Incubated at 37oC for 1 hour. Plated 25μL of 1/100 and 1/1000 dilutions of liquid cultures onto Chloramphenicol LB plates.

Miniprepped liquid culture of Kan BbsA/B' #1 made 30-06-2010.

Ran gel of Enzyme efficiency experiment performed on 30-06-2010.

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Ran gel of digests of Minipreps of ccdB part plasmids performed on 30-06-2010 to ensure they are as expected.

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<--AMANDA miniprep of ccdB BfuA/B', ccdB BfuB/A' part plasmids, digest with NotI?-->

July 2, 2010

Building Parts

The colonies on the 1/100 and 1/1000 dilution plates of ccdB Bbs A/B', ccdB Bbs B/A', cddB BsaA/B', ccdB BsaB/A' part plasmids made on 01-07-2010, were spaced appropriately so as to be able to pick individual colonies. We picked as many colonies as possible and streaked on both LB plates with Kanamycin and Chloramphenicol resistance and LB plates with only Choramphenicol resistance. Again to determine if ccdB was infact inserted.

July 6, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Set up 5ml overnight tubes with 5ml LB broth, 5ul kanamycin, 2.5ul ampicillin from plates made on 30-06-2010. Results: All seven tubes had growth and no growth in control tube.

Experiment continues on 07-07-2010.

July 7, 2010

Building Parts

We made minipreps from the overnights from 06-07-2010. Followed Fermentas protocol to prepare 7 tubes. Restriction Digest with XbaI and PstI Protocol to check for orientation:

    Kan AB in pSB1A3 1ul
    XbaI enzyme 2ul
    PstI enzyme 1ul
    NEBuffer 3 4ul
    MilliQ H2O 3ul
    BSA 4ul
    Total 14ul

Incubated at 37C for 1 1/2 hours.

Gel Electrophoresis:

This is a shared gel. Lanes 2-8 are pSB1A3 plasmids with AB Kan inserts from different tubes, digested with XbaI and PstI. The plasmids from lanes 2 and 3 produced proper fragments. The rest of the tubes were discarded.

Restriction Digest with BsaI Protocol:

    Kan AB in pSB1A3 (247.4 ng/ul or 255.8 ng/ul) 1ul
    chlor or tet AB fragment 1ul
    BsaI enzyme 0.5ul
    NEBuffer 4 3ul
    MilliQ H2O 5.5ul
    Total 11ul

Incubated at 50C for 1 hour.

Ligation Protocol:

    Digested pSB1A3 with Kan AB and chlor or tet 8ul
    T4 DNA ligase 1ul
    5X T4 DNA ligase buffer 6ul
    MilliQ H20 5ul
    Total 20ul

Incubated at room temperature for 1 hour.

Gel Electrophoresis:

Insert gel here.

Experiment continues on 08-07-2010.

July 8, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Followed Inoue transformation protocol using 50ul DH5-alpha competent cells and 2ul of pSB1A3 ligated with tet or chlor made on 07-07-2010. Plated 200ul and 600ul on chlor/amp plates for chlor insert and on tet/amp plates for tet insert. Results:

    chlor/amp plate 1 (200ul): ~300 colonies
    chlor/amp plate 2 (600ul): TNTC colonies
    tet/amp plate 1 (200ul): ~20 colonies
    tet/amp plate 2 (600ul): ~30 colonies

Experiment continues on 09-07-2010.

July 9, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Made overnights with 5ml LB, [20ul chlor and 2.5 ul amp] or [20ul tet and 2.5 ul amp] using transformation plates from 08-07-2010. Results were growth in all experimental tubes and no growth in control tubes.

Experiment continues on 10-07-2010.

July 10, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

We prepared minipreps of tet in pSB1A3 (4 tubes) and chlor in pSB1A3 (7 tubes) from overnights 09-07-2010.

Experiment continues on 12-07-2010.

July 12, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Restriction Digest with EcoRI Protocol to determine plasmid size:

    pSB1A3 with chlor or tet insert 1ul (made on 10-07-2010)
    Fast Digest EcoRI 1ul
    10X Fast Digest Buffer 2ul
    MilliQ H2O 16ul
    Total 20ul

Incubated at 37C for 5 mins.

Gel Electrophoresis:

Lanes 2-5 are pSB1A3 plasmid with tet AB insert. Lanes 6-13 are pSB1A3 plasmid with chlor AB insert. All plasmids were digested with EcoRI. All but the products of lanes 3,6,10,11,12 were correct. 1 out of the 4 tet in pSB1A3 tubes cut incorrectly and was discarded, 4 out of the 7 chlor in pSB1A3 tubes cut incorrectly and was discarded.

Experiment continues on 14-07-2010.

July 14, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Double Digest of chlor, kan and tet in PSB1A3 and amp is pSB1C3 with Xba and PstI protocol:

    plasmid with antibiotic part 1ul
    10X NEBuffer 3 1ul
    MilliQ H20 5ul
    XbaI 1ul
    PstI 1ul
    10X BSA 1ul
    Total 10ul

Incubated at 37C for 1 hour.

Gel Electrophoresis:

Digests of antibiotic AB fragments with XbaI and PstI. Lanes 2-3 are kanamycin AB, lanes 4-6 are chloramphenicol AB, lanes 7-9 are tetracycline AB, lanes 10-12 are ampicillin AB. All of the plasmids cut correctly except for one of the amp in pSB1C3, which was discarded.

Experiment continues on 15-07-2010.

July 15, 2010

Building Parts

Constructing Bsa AB Kan, Chlor and Tet Parts

Antibiotic parts, made on 14-07-2010, are now stored in the DNA box according to the BioBytes registry.

Glycerol stocks were made of overnights from chlor, tet, and kan in PSB1A3 plates.