Team:Aberdeen Scotland/notebook/quorumsensing

=Quorum Sensing Notebook=

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

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

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)
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.
 * 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

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

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

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

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

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

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

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

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

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.