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Team:Aberdeen Scotland/notebook/quorumsensing
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===Day 5 Friday (07/08/09)=== | ===Day 5 Friday (07/08/09)=== | ||
| - | *Team meeting <br><br> | + | *Team meeting |
| + | <br><br> | ||
==Week 10 (10/08/09 - 14/08/09)== | ==Week 10 (10/08/09 - 14/08/09)== | ||
Latest revision as of 15:43, 23 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
- 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
Note: Difficult to see difference between single and double digests for J series promoters (J23105, J23107 + J23115). This should be expected since
all ~30bp in length.
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 - Solving digestion problem (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 (see wet lab)
- 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 alternative 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 (06/07/09 - 10/07/09)
Day 1 Monday (06/07/09)
- Digestion of psB4C5
- in a total volume of 40µl (more concentrated than previous digestion)
- Run gels from today's and Friday's digests (psB4C5, K081008, psB1AT3 + psB1AC3)
- successful digestions - K081008, I0462
- incomplete digestions - psB1AT3, psB1AC3
Concentration of psB4C5 was too low to proceed with cloning
Solution: Perform multiple plasmid preps and combine, check conc. using uv spectrophotometer.
Day 2 Tuesday (07/07/09)
- Multiple plasmid preps of psB4C5.
- Digestion of psB4C5
- Using E + P for double digest
- Purification of I0462 digest (to concentrate)
- Run gels for psB4C5 and I0462
- gel indicates conc. of psB4C5 = 7ng/µl
- gel indicates conc of I0462 = 15ng/µl
Day 3 Wednesday (08/07/09)
- Alkaline phosphatase treat psB4C5
- Calculations for ligation
- Perform ligation
- Transform ligation mixes
- grow overnight on agar plates containing chloramphenicol (50%).
Day 4 Thursday (09/07/09)
Result: Cloning from yesterday yielded unusual results
- many more colonies on controls (vector only and vector + ligase) than on Vector + ligase + inserts.
Conclude: Repeat transformation repeated using 100% chloramphenicol plates and repeat digestion of psB4C5, ensuring complete digestion
Day 5 Friday (10/07/09)
- Perform colony screening
- patch out colonies to identify red colonies (indicate intact vector)
- Repeat digestion of psB4C5
- Run above digestion on gel
- digest appears incomplete (linearised plasmid is still evident in double digest lane)
- Team meeting
Week 6 (13/07/09 - 16/07/09)
Day 1 Monday (13/07/09)
- Repeat digestion of psB4C5
- using alternative PstI
- Run gel
- Results indicate incomplete digestion
- Perform colony screening PCR
- Run PCR gel.
- primer dimers are evident in most lanes, indicating PCR amplification was unsuccessful.
- Repeat digestion of psB4C5
- incubate overnight
Day 2 Tuesday (14/07/09)
- Alkaline phophatase psB4C5
- Repeat ligations (psB4C5 + I0462 + K081008)
- Transformation of ligations
Day 3 Wednesday (15/07/09)
- PCR colony screening
- Run PCR products on gel
- None of the PCR products show presence of expected insert length (~1.6kb)
- Primr dimers are evident in most lanes
Conclude: Create fresh screening plates and perform new PCR
Day 4 Thursday (16/07/09)
- Perform PCR using fresh screening plates
- Run gel of above PCR
- Non of our PCR products yielded expected insert length.
Day 5 Friday (17/07/09)
- Team meeting
Week 7 (20/07/09 - 24/07/09)
Day 1 Monday (20/07/09)
- Perform diagnostic ligation
- using alternative plasmid (psB1AC3) and inserts (I732820 + S03518) known to work.
- Transformation of above ligations
Results: Transformants indicate problem with K081008 (insert 1)
- Many colonies when I0462 (insert 2) cloned with I732820 (insert 1)
- few colonies when K081008 (insert1 )cloned with S03518 (insert 2)
Day 2 Tuesday (21/07/09)
- Repeat transformation of K081008
- Prepare colony screening plates for diagnostic ligations
Day 3 Wednesday (22/07/09)
- Perform colony screening PCR
- Run above PCR products on gel
- results indicate problem with PCR - no amplification, even in positive control lane
Day 4 Thursday (23/07/09)
- Plasmid prep to purify K081008
- Digestion of K081008
- using Roche enzymes
- Run above digest on gel
- gel indicates complete digestion and good concentration
Day 5 Friday (24/07/09)
- Team meeting
Week 8 (27/07/09 - 31/07/09)
Day 1 Monday (27/07/09)
- Calculations for ligations
Day 2 Tuesday (28/07/09)
- Ligation of psB4C5 + I0462 + K081008 (newly transformed and digested)
- Transformation of above ligation
- Grow overnight
- Preparation for beta-galactosidase assay
- Preparation of high competency e.coli (using lac- strain)
- Transformation of above strain with PUC.
Day 3 Wednesday (29/07/09)
- Perform colony screening PCR of yesterday's ligations
- Run PCR products on gel
- PCR was unsuccessful, problems detected as previously seen (22/07/09)
Day 4 Thursday (30/07/09)
- Repeat PCR using new taq polymerase
- Primer dimers detected in all lanes
Conclude: Repeat PCR, check PCR program and perhaps use less colony.
Day 5 Friday (31/07/09)
- Team meeting
Week 9 (03/08/09 - 07/08/09)
Day 1 Monday (03/08/09)
- Repeat transformation of Lac-
- using PRS425.
- Create fresh screening plate for PCR
Day 2 Tuesday (04/08/09)
- Perform PCR
- Check program
- Run PCR products on gel
- PCR problem fixed!
- Expected insert length still not visible
- Inoculate colonies for beta-gal assay
- lac-, lac+, xl1-blue
Day 3 Wednesday (05/08/09)
- Beta-gal assay (chloroform method)
- using lac- and lac+ strains (negative and positive control)
- Repeat using xl1-blue (negative control)
Day 4 Thursday (06/08/09)
- Digest PCR product to identify insert 2
- using HindIII
- Run digest on gel
- results indicate incomplete digestion
- Repeat digestion using Roche enzymes
- Results difficult to interpret.
Day 5 Friday (07/08/09)
- Team meeting
Week 10 (10/08/09 - 14/08/09)
Day 1 Monday (10/08/09)
- Beta-galactosidase assay (inoculations performed 09/08/09)
- using lac-, Lac+ and xl1-blue.
- using sonication to lyse cells
- experimenting with different volumes/concentrations of lysate
Day 2 Tuesday (11/08/09)
- Repeat beta-gal assay (complete run through)
- using sonication to lyse cells
- Normalising lysate samples
- using different ratios for alpha:omega complementation.
- Incubate samples overnight at 4 degrees (5:1, 1:5).
Day 3 Wednesday (12/08/09)
- Perform beta-gal assay using samples incubated overnight.
Day 4 Thursday (13/08/09)
- Make up new Xl1-Blue plates
- Inoculate samples overnight for final beta-gal assay
- lac-, lac+, lac- [PRS425], XL1-Blue [PRS425], Xl1-Blue
Day 5 Friday (14/08/09)
- Final beta-gal assay
- Normalising lysate samples
- using ratios 1:1, 4:1 and 1:4
- + incubation overnight (19 hours)
- Team Meeting
- Team photo
Week 11 (17/08/09 - 21/08/09)
Day 1 Monday (17/08/09)
- Upload Quorum Sensing section to Wiki.
- Work on ethics section
- Prepare graphs from Friday's beta-gal experiment
Day 2 Tuesday (18/08/09)
- Prepare poster template designs
- Prepare beta-gal section for poster
Day 3 Wednesday (19/08/09)
- Upload notebook to wiki
- Add graphics to QS section.
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