From 2010.igem.org

Revision as of 03:14, 22 July 2010

Quorum Sensing Team

Members include Alex Pyden, Marcus Lehr, Jennifer Hong, Eric Raynal, Katie Miskovich, and Audra Williams

7/7/10

Jennifer and Alex - in Lin Lab

Made CaCl₂ stock solution: 0.1M - 50mL

• molar mass = 111 g/mol
• (0.1 mol/L)(.05 L)(111 g/mol) = 555mg
• dissolved 555 mg CaCl₂ in 50mL DIwater
• vacuum filtered into 50mL tube

Made ampicillin stock solution:

 -1 g ampicillin
 -5mL DIwater
 -ethanol

• filtered by syringe into 15mL tube
• put into ten 1mL alliquots in 1.5mL tubes
• stored in -20°C in ERB lab

Inoculated E. coli DH5α from frozen stock into 12 mL LB broth

• put in 30°C, 200 rpm shaking, overnight

~1.5 hrs

7/8/10

Alex, Jennifer and Eric - in ERB

Made LB agar plates w/ ampicillin

 -10 g LB broth
 -7.5 g agar
 -500 mL DI water

• autoclaved mixture at 121°C for 30 min (sterilize time), following Mike Nelson's protocol

Created protocols for Obtaining Deionized Water in the ERB and ERB Spectrophotometer

• uploaded to Team:Michigan/Protocols section

poured 20 LB-amp plates (large)

• left to solidify in ERB 1230
• 12 plates were used by Ann for biobrick transformations
• 8 plates were stored in 4°C ERB

~3.5 hrs work

Ann, Marc, Audra and Katie - in Lin Lab

Performed biobrick transformations using heat shock

• Followed protocol for Transformation-heat shock
• protocol located under DNA Manipulation in Team:Michigan/Protocols section

After second washing:

• ODs for sample 1 and 2 were 0.378 and 0.358 respectively

4 hrs of work

7/17/10

Alex & Jennifer - in ERB

Yesterday, Marcus stored newly obtained strains in 4°C in ERB

• W3110 w/ plasmids pTC6 & pET-GFP
  • AI-2 reporter (E. coli, amp and kan-resistant)
• MDAI2 w/ plasmids pCT6 & pET-GFP
  • AI-2 reporter & LuxS null mutant (E. coli, does not produce AI-2, amp and kan-resistant)

Removed these strains from 4°C -- they are stored in soft agar stab cultures Made one streak plate on LB+amp for each strain from stabs

• placed in 374°C (rm. 1239)

Made one 2mL LB+amp broth cultures in a 15mL tube for each strain from stabs

• placed in 30°C, 200 rpm shaking (rm. 1230)

Placed agar stabs and LB+amp broth in 4°C

~1.5 hrs work

Created protocol/plan for experiment. Testing AI-2 Response

--Infekt 00:26, 18 July 2010 (UTC)

7/19/10

Alex and Marcus

7/17 plates are no good -- they needed kanamycin

• plates were discarded
• broth cultures had kanamycin added a day later -- too late to be cryostored, but they were transfered to ERB 4°C

Made LB agar

-20 g powder/500 mL DI water

• autoclaved 30 min (sterilize time) 255°C
• poured 4 LB plates
• poured 6 LB+amp plates (100 μg/mL ampicillin)
• poured 10 LB+amp+kan plates (50 μg/mL kanamycin)
• all plates left to cool in ERB 1230

Obtained a rotor for 1.5mL tubes for the centrifuge from Rodger Pinto

• placed in centrifuge in cold room ERB 1224

Obtained LuxS (strain JW2662-1) & MarC (for Jeremy Minty) null mutant E. coli strains from [http://cgsc.biology.yale.edu/ CGSC]

• retrieved from Lin 4°C
• made one spread plate on LB for each strain following [protocol]
• placed both plates in ERB 37°C

Transfered iGEM cryobox from Lin Lab -20°C to ERB -20°C

~6 hrs

--Infekt 23:49, 19 July 2010 (UTC)----

7/21/10

Alex, Eric and Jeremy

{Yesterday, Marcus made broths for E. coli JW2662-1 (LuxS^-), E. coli JW1522-1 (MarC^- for Jeremy Minty), E. coli W3110, E. coli MDAI2, P. putida for Oil Sands and P. fluorescens for Oil Sands

• 2 mL LB each culture}

Crystored all six strains following Making frozen stocks protocol

• stored in iGEM box -- -80°C Lin Lab

Plated LuxS^- and MarC^- mutants and Pseudomonas strains on LB Plated W3110 and MDAI2 on LB+amp+kan

• placed all six plates in 37°C ERB 1239

Stored remaining clean LB+amp (6) and LB+amp+kan (8) plates from 7/19 in 4°C ERB 1239

~1.5 hrs

--Infekt 03:14, 22 July 2010 (UTC)