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Team:Aberdeen Scotland/notebook/quorumsensing
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===Day 1 Monday (29/06/09)=== | ===Day 1 Monday (29/06/09)=== | ||
Revision as of 11:11, 22 August 2009
University of Aberdeen - Pico Plumber
iGEM 2009
Quorum Sensing Notebook
Week 1 - Research and Biobrick Rescues (08/06/09 - 12/06/09)
Day 1 Monday (08/06/09)
- Researching the Registry for Biobricks
- Identified K081008, J23100, C0062, K091156, B0034,B0030, B0015, R0061 and R0062 for possible use in our project.
- Other biobricks identified for other sub-projects include R0011.
Day 2 Tuesday (09/06/09)
- Researching the literature for information on Quorum Sensing.
- Researching for information on LVA tags
Day 3 Wednesday (10/06/09)
- Preparing for biobrick rescue and transformation
- Prepare LB and Agar
- Prepare Cacl2 solution,
- Prepare 1M MgCl2, 1MMgSO4 and 2M glucose solutions (for SOC medium)
- Inoculate e.coli and grow overnight
Day 4 Thursday (11/06/09)
- Rescue Biobricks (K081008, J37033, B0015, J23100, R0011, R0062, K112808, C0051, C0040, I732100, R0051, K145001 and B0034)
- Prepare competent e.coli, using calcium chloride method
- Transform e.coli with biobricks listed above
- grow overnight on agar/amp plates.
Day 5 Friday (12/06/09)
- Results from yesterday indicate that only I732100 was successfully transformed into e.coli
- Identify possible recipient plasmids for QS module (psB4C5).
- Propose a cloning strategy
- Team meeting
Week 2 - Biobrick rescue and purification (15/06/09 - 19/06/09)
Day 1 Monday (15/06/09)
- Inoculation of transformants - B0015, I732100, R0011 and K081008.
Day 2 Tuesday (16/06/09)
Inspection of inoculations, after overnight growth, revealed, as suspected, that only I732100 had grown
- Perform plasmid prep on I732100
- Digest I732100
- 1 Double digest E+ S
- 4 single digests - E, S, X and P
- Prepare TSS buffer, for TSS method of competency (to be performed tomorrow)
- Inoculate e.coli and grow overnight for tomorrow's TSS method.
Day 3 Wednesday (17/06/09)
- Rescue additional biobricks - K112022, R0040, I732094, K093005, E0840
- Prepare competent e.coli using TSS method.
- Transform with biobricks rescued today + B0015, R0011, R0051, K112808 and K081008, grow overnight
Day 4 Thursday (18/06/09)
- Prepare for miniprep
- inoculate transformed e.coli and grow overnight in liquid medium
- Create master plates to keep record of colonies used
Day 5 Friday (19/06/09)
- Perform miniprep to purify R0011, R0051, B0015, K112808, K081008
- Team meeting
Week 3 - Digestions (22/06/09 - 26/06/09)
Day 1 Monday (22/06/09)
Day 2 Tuesday (23/06/09)
- Transformation of ccdB resistant e.coli with plasmids psB3C5, psB3K5, psB3T5, psB4C5, psB4T5 and psB4K5.
- Digestion of purified biobricks - (R0011, C0051, I732094, B0015, E0840, R0040, K112022, K112808, K093005, R0051, K081008, J37033, R0062 and I732100)
- Perform gel electrophoresis of today's digests
- unsuccessful - C0051+ R0062 + B0015 + R0040 + E0840 (double digest problem), J37033(empty lane), I732100 (unusual bands),
- Successful - C0040, K145001
Day 3 Wednesday (24/06/09)
- Digestion of lysis cassettes- K112022 + K112808
- Using BamHI and BglI
- Repeat digestion of I732100
- Double digests - E + S and X + P
- Single digests - E, S, X and P
- Run gels for above digestions
- Results indicate digestion needs to be repeated
- Inoculate trasnformed e.coli and grow overnight
Day 4 Thursday (25/06/09)
- Plasmid prep to purify biobricks - B0030, I0462, J23105, J23107, J23115, S03518, psB4K5 and psB3T5.
- Digestion of above plasmid preps, plus additional C0051, psB1AC3 + psB1AT3
- Perform gel electrophoresis of above digestions (minus J23107, J23105 and J23115)
- successful digestions - psB3T5 and psB1AT3
- unsuccessful digestions - psB1AC3, psB4K5, B0030 and C0051.
- Results also indicated that double digests using X + P were unsuccessful and should be repeated- S03518 and I0462
Day 5 Friday (26/06/09)
- Digestion of K081008
- double digest using E + S
- Single digests using E
- Perform gel electrophoresis for K081008 digest
- Results indicate incomplete digestion.
- Team meeting
Week 4 29/06/09 - 03/07/09
Day 1 Monday (29/06/09)
- Digestion of lambda DNA
- using HindIII
- Repeat digestion of E0840
- using alternative digestion protocol
- dilution of single digest prior to second enzyme addition
- using alternative digestion protocol
- Run gels for above digests
- gel indicates that alternative digestion method eliminates double digest problems seen previously
- Inoculate I0462, psB4C5 and psB3K3 from master plates
Day 2 Tuesday (30/06/09)
- Perform plasmid prep to purify psB3K3, psB4C5 + I0462.
- Digestion of above plasmid preps
- using dilution method
- Run gels using above digests
- Gels indicate that I0462 and psB4C5 were successfully digested, psB3K3 yielded empty lanes
Day 3 Wednesday (01/07/09)
- Perform transformation using psB3C5
- Inoculate K081008, psB1AT3 and psB1AC3
- No overnight growth was detected for K081008 - repeat transformation
Day 4 Thursday (02/07/09)
- Research primer ordering
- Transformation of K081008
Day 5 Friday (03/07/09)
Perform plasmid prep to purify K081008, psB1AT3 and psB1AC3 Digestion of above plasmid preps
- using E + P double digests for plasmids and E + S for K081008.
- Team meeting
Week 5 05/07/09 - 10/07/09
Week 6 13/07/09 - 16/07/09
Week 7 20/07/09 - 24/07/09
Week 8 27/07/09 - 31/07/09
Week 9 03/08/09 - 07/08/09
Week 10 10/08/09 - 14/08/09
Week 11 17/08/09 - 21/08/09
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