From 2010.igem.org

Revision as of 23:12, 26 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 Culturing CGSC Strains 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)---- Update: --Infekt 03:03, 23 July 2010 (UTC)

7/21/10

Alex, Eric and Jeremy

{Yesterday, Marcus made broths for E. coli JW2662-1 (LuxS^-) (from spread plate), E. coli JW1522-1 (MarC^- for Jeremy Minty) (from spread plate), E. coli W3110 (from stab), E. coli MDAI2 (from stab), 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)

7/22/10

Alex

During Lab Committee meeting -- Moved the six plates from yesterday from 37°C to 4°C

Uploaded Protocol: Culturing CGSC Strains

--Infekt 03:04, 23 July 2010 (UTC)

7/23/10

Alex

Updated Strain Database --Infekt 00:22, 24 July 2010 (UTC)

7/25/10

Alex and Eric

Made 250 mL LB broth in each of two 500mL flasks

-5 g LB broth
-250 mL DI water

• autoclaved 30 min (sterilize time) ~255°C

Autovclaved 250 mL DI water in each of two 500mL flasks

• 30 min sterilize time, ~255°C

Made a 2mL culture in 15mL tube of W3110

-2 mL LB from 4°C
-2 μL 100mg/mL ampicillin (final conc. 100μg/ml)
-2 μL 50mg/mL kanamycin (final conc. 50μg/mL)

• incubated overnight, 30°C, 200 rpm shaking -- 1pm

Left stuff in autoclave

--Infekt 17:49, 25 July 2010 (UTC)

7/26/10

Alex and Marcus

{yesterday, Josh and Charlie removed stuff from the autoclave}

Analyzed yesterday's W3110 culture on Lin microplate reader

• endpoint - cuvette - Ex 450nm, Em 520nm, cutoff 515nm (GFP settings)
  • blank: 91.591 RFU
  • W3110: 117.47 RFU
• fixed Ex 450nm - Em spectrum scan - cuvette
  • W3110 - Em peak = 520nm (green - good)
• fixed Em 520nm - Ex spec scan - cuvette
  • W3110 - Ex peak = 370nm (wtf) - still good emission at Ex 450nm
• fixed Ex 370 - Em spec scan - cuvette
  • W3110 - Em peak = 450nm (wtf)
• endpoint - cuvette - Ex 370nm - Em 450nm
  • W3110: 1054.9 RFU
  • blank: 1934.6 RFU
    • This is some kind of background - very high readings, but the blank is higher than the sample - maybe the GFP in the sample is reabsorbing at 450nm?? At any rate, there appears to be GFP, as expected, and we can probably use Ex 450nm and Em 520nm to detect it.

Autoclaved two 500mL flasks to be sterile containers, each with ~150 mL DI water inside

• 30 min sterilization (55min total), ~250°C

Alex

Learned how to use Epifluorescence Microscope in HHDow (2nd floor) from Alissa

• Viewed yesterday's W3110 culture for GFP
  • Unfortunately, no fluorescence could be detected. However, GFP was presumably detected using the microplate reader, so maybe it was just at a very low level. Maybe, the culture was too old (~24 hrs). We will try again tomorrow with a 16-hr culture.
• Uploaded Epifluorescence Microscope Usage protocol

Removed glassware from the autoclave --> ERB 1230 Made broth cultures for W3110 and MDAI2

• each 5 mL LB + 50μg/mL kan + 100μg/mL amp in a 15mL tube (added 5 μL each of a 1000X stock of each antibiotic)
• incubated 30°C, 200 rpm shaking -- 6:30 pm