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
• 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 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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