Team:Michigan/Alex's Notebook

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Alex's Wiki

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

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 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

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, idk if these have GFP or what. At any rate, if they do, the MDAI2 is too close to W3110. We'll probably ahve 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

8/4/2010

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:

well	pellet	supernatant
A1	MDAI2	LB
A2	MDAI2	MDAI2
A3	MDAI2	W3110
A4	MDAI2	C. vulgaris
B1	LuxS^-+pLsrA-YFP	LB
B2	LuxS^-+pLsrA-YFP	MDAI2
B3	LuxS^-+pLsrA-YFP	W3110
B4	LuxS^-+pLsrA-YFP	C. vulgaris
D1	[blank]	LB
D2	[blank]	MDAI2
D3	[blank]	W3110
D4	[blank]	C. vulgaris

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

8/19/10

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

--Infekt 04:08, 20 August 2010 (UTC)

8/20/10

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

8/22/10

Alex

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

--Infekt 19:21, 22 August 2010 (UTC)

Made BL21 broth culture

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

--Infekt 02:04, 23 August 2010 (UTC)

8/26/10

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

--Infekt 04:12, 27 August 2010 (UTC)

8/27/10

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:

well	pellet	supernatant
A1	MDAI2	LB
A2	MDAI2	W3110
A3	MDAI2	BL21
A4	MDAI2	C. vulgaris
B1	LuxS^-+pLsrA-YFP	LB
B2	LuxS^-+pLsrA-YFP	W3110
B3	LuxS^-+pLsrA-YFP	BL21
B4	LuxS^-+pLsrA-YFP	C. vulgaris
C1	[blank]	LB
C2	[blank]	MDAI2
C3	[blank]	W3110
C4	[blank]	C. vulgaris

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

Returned supernatants to ERB -20°C --Infekt 00:50, 28 August 2010 (UTC)

8/28/10

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.

--Infekt 17:25, 28 August 2010 (UTC)

9/12/10

Alex

Made E. coli K12 broth culture from plate

2 mL LB in 15mL tube
37°C, 200 rpm shaking - ERB 1230

Uploaded NanR cloning protocol

--Infekt 05:35, 13 September 2010 (UTC)

9/29/10

Alex and John

Dissolved M9 salts to 50X

All of component A in 1000 mL DIwater
All of component B in 1000 mL DIwater

Autoclaved both: each split into two 1000mL bottles filled w/ 500mL each (four bottles total)

30 min sterilize time

Began biofilm assay following Alex's biofilm assay protocol

Made broth cultures of P. putida LD1, P. fluorescens LD2, and LD1+LD2 coculture from plates, and P. putida KT2440 from Lin Lab frozen stock

KT2440 inoculated into 2 mL LB in 15mL tube
others inoculated into 2 mL LB + 100μg/mL amp in 15mL tube
all incubated in 30°C, 200rpm shaking -- ERB 1230, 8:45pm

Streaked KT2440 on LB plate from frozen

incubated 37°C -- ERB 1239

Added KT2440 to strain database Mixed M9 A and B solutions and diluted 1/50 in 200 mL sterile DIwater -- in 4 50mL tubes

each tube 1 mL M9A, 1 mL M9B and 48 mL water

Alex

Uploaded Alex's biofilm assay protocol

Added borax to two of the M9 tubes (100 mL) to buffer at ~pH 9.2

- (.01 mol/L)*(.05 L)*(381.37 g/mol) = 190.7 mg borax/50 mL water

Tested pH with pH indicator strips
- ~9
Vacuum-filtered 100 mL w/ steriflips

9/30/10

Alex

Aliquot M9 and buffered M9 into five 15mL tubes each Need 15 mL stocks of each of the following media -- each 15 mL neutral and 15 mL buffered at pH 9:

M9
M9 + 0.4% glucose
M9 + Acros NAs (250mg/L)
M9 + Sigma Aldrich NAs (250mg/L)
M9 + both Acros & Sigma NAs (each 250mg/L)
LB

Added 150 μL 40% glucose to appropriate tubes (.4% final concentration) Aliquot 15 mL LB into each of two 15mL tubes Added NAs to appropriate tubes

- Acros: (.25 mg/mL)*(15mL)/(.91 mg/μL) = 4.12 μL
- Sigma: (.25 mg/mL)*(15mL)/(.92 mg/μL) = 4.08 μL

added 4.1 μL of each NA to each respective tube

Added borax to one tube of LB to buffer at pH9

- (190.7 mg)*(15μL/50μL) = 57.2 mg per 15mL LB

tested pH with indicator strip
- ~9

Tested pH of neutral M9 with indicator strip

~7

Moved KT2440 LB plate from 37°C to 4°C -- ERB 1239

Continued Alex's biofilm assay protocol

made one 96-well plate for all strains and media at pH7 only
plate template :

- 100 μL/well: 99 μL medium + 1 μL culture
- covered plate with gas-permeable membrane
Started 24-hr kinetic reading in Lin Lab microplate reader
- 30°C
- OD600 absorbance
- read every 30 min

Made broth cultures for LD1, LD2 and KT2440 from yesterday's overnight broths

2 mL each in a 15mL tube
30°C, 200 rpm shaking - 6:20pm

Discarded yesterday's overnight broths

--Infekt 00:57, 1 October 2010 (UTC)