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Team:Alberta/Notebook July
From 2010.igem.org
July 2010
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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.
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.
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.
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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.
<--Image w/ explanation-->Ran gel of digests of Minipreps of ccdB part plasmids performed on 30-06-2010 to ensure they are as expected.
<-- Image w/ explanation--><--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
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.
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.July 8, 2010
Building 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
July 9, 2010
Building 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.
July 10, 2010
Building Parts
We prepared minipreps of tet in pSB1A3 (4 tubes) and chlor in pSB1A3 (7 tubes) from overnights 09-07-2010.
