Team:Aberdeen Scotland/notebook/quorumsensing

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University of Aberdeen iGEM 2009


Contents

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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