Quorum Sensing Team

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

7/7/2010

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

7/8/2010

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

7/17/2010

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 37°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

7/19/2010

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

7/21/2010

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

7/22/2010

Alex

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

Uploaded Protocol: Culturing CGSC Strains

7/23/2010

Alex

Updated Strain Database

7/25/2010

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

7/26/2010

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

7/27/2010

Alex, Eric and Marcus

Checked OD600 of yesterday's cultures in Lin Lab spectrometer

• W3110: 0.873
• MDAI2: 0.855

Need to start culture with OD .02

• .02 / .873 = 2.29%
• .02 / .855 = 2.34%

Obtained 40% glucose solution from Ann - Lin Lab

• need .8% glc final conc.
• .8/ 40 = 2%

Started 100mL culture of W3110 in 500mL flask

-2.29 mL W3110 culture
-2 mL 40% glucose
-95.7 mL LB

• • 100 - 2.29 - 2 = 95.71

Started 100mL culture of MDAI2 in 500mL flask

-2.34 mL MDAI2 culture
-2 mL 40% glucose
-95.7 mL LB

• • 100 - 2.34 - 2 = 95.66
• placed both cultures in 37°C, 225 rpm shaking - ERB 1230, 11:50am

Marcus and Eric

Alex and Marcus

Checked remaining MDAI2 and W3110 cultures on fluroescence microscope in LSI 6th floor

• both seem to fluoresce at GFP equally
  • This is probably not right; MDAI2 should not have GFP. Will check on a fluorospectrometer tonight

Alex

Read W3110 & MDAI2 cultures on microplate reader in Xi Lab - SPH

• Ex 485nm, Em 545nm (closest to GFP possible on this reader)
  • W3110 = 6
  • MDAI2 = 5
  • Many Acinetobacter controls were run - all at 0 or 1
  • This is bad -- MDAI2 is clearly producing GFP, meaning it produces AI-2. Need to contact Tsao authors.

7/28/2010

Alex

Made 40 mL of 0.1% crystal violet solution in Xi Lab - SPH

• for Ann; will deliver tomorrow

7/30/2010

Alex and Marcus - w/ Charlie and Prae

Performed transformation of 5 parts following Transformation-electroporation protocol -- starting from reading OD of overnight. (Ann started the previous steps.)

• read OD of overnights (in Lin Lab)
  • JW2662-1 (LuxS^-): 1.138 -- bad
  • DH5α: 0.710 -- good
• made a 1:2 dilution of JW2662-1:LB (48 mL total)
  • placed in 30°C, 200 rpm shaking, 30 min
  • removed and read OD
    • JW2662-1: 0.657 -- good
• Transfered entire 47 mL of each culture to a 50mL tube

Continued with protocol...

• following spins and washes, read OD again:
  • JW2662-1: 0.42 -- good
  • DH5α: 0.87 -- good
• electroporated:
  • into DH5α:
    • pBAD (for Ann)
    • INP-GFP (for virus group)
    • INP-linker (for virus group)
    • OmpA-GFP (for virus group)
  • into JW2662-1
    • pLsrA-YFP (two sources: L14 and 008)
  • time constants:
    • OmpA-GFP: 2.8
    • INP-GFP: 2.8
    • INP-linker: 3.2
    • pLsrA-YFP (008): 5.6
    • pLsrA-YFP (14L): 5.6
    • pBAD: 4.8
    • neg. control: 5.0
  • incubated all 30°C -- 4:30pm to 7:30pm - 3 hrs
• plated on:
  • OmpA-GFP: LB+amp
  • INP-GFP: LB+amp
  • INP-linker: LB+amp
  • pLsrA-YFP (both): LB+amp+kan
  • pBAD: LB+kan+IPTG
  • control: LB+amp AND LB+kan+IPTG
• placed all plates in 37°C
• excess electroporation culture stored in 4°C

8/2/2010

Alex and Eric

Read overnight cultures on Lin Lab spectrometer

• OD600 (endpoint)
  • LB: 1.156
  • W3110: 1.100
  • MDAI2: 1.156
• GFP fluorescence (Ex 450nm, Em 520nm; endpoint)
  • LB: 90.291 (RFU)
  • W3110: 190.59
  • MDAI2: 180.86
• Em 520nm, Ex sweep
  • LB: peak 370nm (some kind of background)
  • W3110: peak 370nm, kinda bimodal at 440nm (GFP)
  • MDAI2: peak 370nm, bimodal at 430nm (GFP)
• Ex 450nm, Em sweep
  • LB: peak 515nm......wtf
  • W3110: peak ~525nm - ok, GFP
  • MDAI2: peak ~525nm - GFP
• Ex 440, Em sweep
  • W3110: peak ~520nm
• Ex 440nm, Em 520nm (endpoint)
  • • • LB: 117.61
  • W3110: 204.86
  • MDAI2: 198.34

OK, I don't know if these have GFP or what. At any rate, if they do, the MDAI2 is too close to W3110. We'll probably have to work with the YFP biobrick instead.

Got C. vulgaris from Bobby Levine

• ~200 mL
• strain 258, from flask 085
• spun 4200 rpm for 10 min
• filtered according to
• stored -20°C ERB 1239

Alex and Marcus

Made broth cultures in 15mL tubes from plates

• 3 mL LB + LuxS^-
• 3 mL LB + 100μg/mL amp + [DH5α + pLsr-YFP]
• 3 mL lB + 100μg/mL amp + 50μg/mL kan + [LuxS^- + pLsr-YFP]
  • placed all in 30°C, 200 rpm shaking
  • moved plates back to 4°C

Autoclaved waste flasks

• 55 min (30 sterilize)
• 255°C

8/3/2010

Alex and Marcus

Took yesterday's overnights to Alex's (Xi) lab since the Lin lab fluorospectrophotometer was in use.

• Excitation Frequency: 485
• Emission Frequency: 545
• Sensitivity: 50

Fluorescence/OD600:

• LuxS^-: 2263
• DH5α + pLsr-YFP: 2078
• LuxS^- + pLsr-YFP: 1456

Very confusing results, LuxS^- was supposed to be the negative control, and shows the highest fluorescence per OD. Some factor must be confounding our assay.

Also, made 10 mL overnights of LuxS^- (pLsr-YFP) and MDAI2 (pET6 + pET-GFP) in 50 mL tubes at 7 pm in LB+Amp+Kan. Put in shaker at 30°C.

8/4/2010

Alex and Marcus

Made broths for experiment tomorrow

• 2 mL LB + 100μg/mL amp + 50μg/mL kan in each of four 15mL tubes - from yesterday's broth cultures
  • MDAI2
  • LuxS^-+pLsrA-YFP
• 2 mL LB + 50μg/mL kan and LuxS^- in a 15mL tube - from 7/19 spread plate
• 2 mL LB + 100μg/mL amp and DH5α+pLsrA-YFP - from 7/13 streak plate
• Incubated all 30°C, 200 rpm shaking - 7:20pm

Made stocks of LB + 50μg/mL kan (25 mL) and of LB + 100μg/mL amp + 50μg/mL kan (50 mL)

• each in a 50mL tube
• stored in 4°C

Updated QS experimental protocol

8/5/2010

Alex

Continued testing AI-2 response of yesterday's cultures, following Lsr Circuit Test Protocols

• Obtained cultures of MDAI2 and LuxS^- from ERB 1230 at 11:30am (16hrs)
• obtained supernatants:
  • MDAI2 (4 hr)
  • W3110 (4 hr)
  • C. vulgaris (one aliquot)
• moved all to Xi Lab, SPH -- cultures to 4°C

Followed protocol

• OD & GFP/YFP readings (OD600, Ex485/Em545): here
  • Similar results with non-GFP strains from Xi Lab. So basically, there is no significant fluorescence in any of these - not even the "positive control" DH5α with pLsrA-YFP. Hopefully this exp works, or it hopefully it will with the new +cntl strain we hope to have soon.

Continued protocol...

• moved plate to RT (bench)

Continued Protocol

• combined pellets & respective supernatants
• plate template:

• Covered plate w/ gas-permeable membrane
• Started reading in Xi microplate reader
  • kinetic; OD600 and Ex485/Em545; 6 hrs at 10min intervals
  • 30°C; shaking for 30sec before each reading

Attended Lab Committee meeting

Researched Heidelberg team

uploaded notebook

made presentation for tomorrow

Finished exp by protocol

• processed data and uploaded it
• discarded 15mL tubes
• discarded both 24-well plates

8/19/2010

Alex

Acquired new strains (soft agar stabs from Tsao Lab)

• W3110 + pTC6 + pET-GFP
• MDAI2 + pTC6 + pET-GFP
• BL21 + pTC5 + pET-GFP
  • produces more AI-2 than W3110
• moved all to ERB 4°C

Made broth cultures of MDAI2 and BL21 from stab cultures

• each 2 mL LB in a 15mL tube
• 37°C, 200 rpm shaking
• placed in incubator at 8pm

Added Quorum Sensing project description to Wiki

8/20/2010

Alex

[Earlier, Marcus cryostored BL21 and MDAI2 in Lin -80°C and made a spread plate of new MDAI2 strain.]

Made streak plate of BL21

• placed in 35°C incubator

Made frozen stock of BL21 and MDAI2

• each 500 μL culture + 500 μL 50% glycerol
• stored in ERB -20°C

Updated strain database

8/22/2010

Alex

Moved MDAI2 spread plate and BL21 streak plate from 37°C to 4°C

Made BL21 broth culture

• 10 mL LB+100μg/mL amp+50μg/mL kan in a 50mL tube
• 37°C, 200 rpm shaking
• 8:00pm

8/23/2010

Marcus

Measured OD600 of overnight in Gulari Lab spectrophotometer.

 -Used LB+ Amp(100μg/mL)+ Kan(50μg/mL) blank
 -OD was 1.55

Made dilution to OD 0.02 into 50 mL flask using OD600 Cell Dilution Protocol.

 -48.3 mL LB
 -1 mL 40% glucose solution
 -0.645 mL overnight culture

Put in 37°C shaker for 4 hours and then collected supernatant according to Lsr Circuit Test Protocols. Supernatant was labeled and stored in the ERB -20°C.

8/26/2010

Alex and Marcus

Made cultures of MDAI2 + pTC6 + pET-GFP (new) and LuxS^- + pLsrA-YFP

• each 2 mL LB + 100μg/mL amp + 50μg/mL kan in each of four 15mL tubes (8 tubes total)
• incubated 30°C, 200 rpm shaking, 9:15pm

8/27/2010

Alex

Obtained cultures and supernatants from ERB -- 1:15pm (16 hrs); transfered to Xi Lab, SPH

Began Quorum Sensing experiment again, following posted protocol

• Used BL21 supernatant instead of MDAI2
• had to wait until 6:45pm to run exp; cells were overgrown and started to die/pellet
  • therefore, took only broth, avoiding pellet, for OD and spin
• spun at 5000rpm instead of 4200
• plate template:

• ran growth curve for 16 hrs (overnight) instead of 6, drawing from Singapore protocol

Returned supernatants to ERB -20°C

8/28/2010

Alex

Obtained data from QS main experiment

• uploaded here

Only the wells with LB added to pellet had a significant increase in fluorescence over time.

• This doesn't make sense, but seems to replicate earlier results.

It seems that the LB blank well was contaminated slightly. This shouldn't really matter, unless the contamination was also in the other two LB wells, in which case maybe co-culture was what cause the increase in fluorescence. However, OD also only increased significantly for the LB wells, so this is probably why the fluorescence also increased. This does present another experiment idea, however: maybe we can try to culture MDAI2 alone, and then in co-culture with K12 to see if there it a difference in GFP. At any rate, I'm not sure it's feasible to co-culture with algae, so quorum sensing project is probably done.

In the Lab