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Team:Alberta/Notebook June
From 2010.igem.org
June 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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June 1, 2010
Building Parts
KanA/B' and KanB/A' fragements PCRed on 11-05-2010, digested with BsaI-HF at 37oC for 1.5hours, heat inactivated at 65oC for 30 minutes. Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other. Also tried to ligate KanA/B' fragments with KanB/A'. Ligated with T4 DNA ligase for 3 hours at 21oC.
Set up liquid cultures of KanRA/B'-Bsa and KanR B/A'-Bsa in pSB1C3 from plates streaked with on 30-05-2010.
June 2, 2010
Building Parts
Miniprepped liquid cultures from 01-06-2010. Ran a 1% agarose gel of the ligations performed 01-06-2010.
To optimize the Restriction and ligation of BsaI-HF, digested KanA/B' and KanB/A' fragements PCRed on 11-05-2010 with the following recipe:
Digestion Recipe:
- 14μL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/μL)
- 5μL 1/10 dillution of 100X BSA
- 5μL 10X NEBuffer4
- 1.5&mu:L BsaI-HF
- 24.5μL MilliQ
Digested at 50oC for 1hour, heat inactivated the enzyme at 65oC for 20 minutes PCR purified the digests.
June 3, 2010
Building Parts
Tried to ligate the KanA/B' fragments to itself. Tried to ligate the KanB/A' fragments to itself. Tried to ligate the KanA/B' fragments to the KanB/A' fragments.
Ligation Recipe:
- 8μL of digest from 02-06-2010 (either 8μL of one of the fragments or 4μL of each)
- 1μL T4 DNA ligase
- 6μL 5X Buffer
- 15μL MilliQ H2O
Took aliquots of ligations at varying times and ran 1% agarose gels to test ligation.
Digested some of Minipreps of the KanA/B' fragment inserted into pSB1C3 from 02-06-2010.
Digestion Recipe:
- 14μL plasmid (approx 300-400ng/&mu:L)
- 5μL 10X NEBuffer 4
- 5μL 1/10 dilution of 100X BSA
- 1.5μL BsaI-HF
- 24.5μL MilliQ H2O
Made a 1/100 dilution of AmpR and TetR PCR Products from 27-05-2010 and performed PCR reactions to produce antibiotic inserts to make parts.
PCR Recipe:
- 35.5μL MilliQ H2O
- 1μL 10 uM dNTPs
- 5μL 10X PCR Buffer
- 2μL 50 uM MgCl2
- 2.5μL Primer + (ApR 1/10+ or TR 1/10+)
- 2.5μL Primer - (ApR 1/10- or TR 1/10-)
- 1μL Template (1/100 diluted AmpR or TetR)
- 0.5μL Taq Polymerase
PCR Program:
- 5 min-94oC
- 45 sec-94oC
- 1 min-60oC
- 1 min-72oC
- Repeat 2 through 4 35 times
- 5 min-72oC
June 4, 2010
Building Parts
Tried to test the limits of ligation reaction. Ligation Recipe of plasmids cut on 03-06-2010:
- 8μL digestion mixture
- 6μL 5X T4 ligase buffer
- 1μL T4 ligase
- 15μL MilliQ
Tried to further reaction of KanA/B' fragments to KanB/A' fragments. To the existing Ligase reactions from 03-06-2010 added:
- 1μL T4 ligase
- 6μL 5X T4 ligase buffer
- 23μL MilliQ
Tried to set limits of Kan fragments that would ligate:
- 24μL digestion from 02-06-2010 (either 24μL of one of the fragments or 12μL of each)
- 6μL 5X T4 ligase buffer
- 1μL T4 ligase
June 8, 2010
Testing Parts
To test the function of E.coli smell variant experiments for our kit, BBa_J45120 (Wintergreen) and BBa_J45200 (Banana) from the MIT 2006 BioBrick Registry were transformed in DH5α cells.
June 9, 2010
Building Parts
Restriction Digested AmpR and TetR inserts from 03-06-2010 to be ligated with psB1C3 vector later on.
Digestion Recipe for AmpR:
- 13.4μL MilliQ H2O
- 0.60&mu:L AmpR (333.3 ng/μL, determined by nanodrop)
- 2μL 10X BSA
- 2μL 10X Buffer 4
- 2μL BsaI
Digestion Recipe fro TetR:
- 13.3μL MilliQ H2O
- 0.7&mu:L TetR (571.3 ng/μL, determined by nanodrop)
- 2μL 10X BSA
- 2μL 10X Buffer 4
- 2μL BsaI
Both digestions were incubated at 50oC for 1hour, heat inactivated the enzyme at 70oC for 20 minutes.
June 10, 2010
Building Parts
Double Digested Kan/Chlor minipreps to determine orientation.
Digestion Recipe:
- 5μL 10X Buffer 3
- 1μL XbaI
- 1μL PstI
- 5μL Kan/Chlor fragments
- 33μL MilliQ H2O
- 5μL 10X BSA
Incubated at 37oC for 1hour, heat inactivated the enzyme at 80oC for 20 minutes.
<---gel image of 10.06.10 Karina1--->Restriction Digested A/B' psB1C3 vector.
Digestion Recipe:
- 10μL MilliQ H2O
- 5&mu:L psB1C3
- 2μL 10X BSA
- 2μL 10X Buffer 4
- 1μL BsaHF
Incubated at 37oC for 1hour, heat inactivated the enzyme at 80oC for 20 minutes.
Ligated digested A/B' psB1C3 with AmpR from 09-06-2010.
Ligation Recipe:
- 9μL A/B' psB1C3 vector
- 5μL AmpR insert
- 6μL 5X Ligase Buffer
- 1μL Ligase
- 9μL MilliQ H2O
Incubated at room temperature for 45 minutes. Transformed with DH5α cells using 15μL of the ligated A/B' psB1C3 with AmpR.
June 11, 2010
Building Parts
To test the function of E.coli smell variant experiments for our kit, BBa_J45120 (Wintergreen) and BBa_J45200 (Banana) from the MIT 2006 BioBrick Registry were transformed in DH5α cells.
