SBB11Ntbk-Team Butterfish

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Mary Wang 22:39, 1 April 2011 (EDT)

Goal:

To evaluate how various stress promoters respond to stress caused by the toxic gene violacein. In essence, we will first transform P_bad-violacein in the cell, then transform a stress promoter-GFP part in the cell, add arabinose until to induce violacein expression, and finally measure the GFP expression on the TECAN plate reader.


Procedural Timeline:

Day 1 (Tuesday, 4/5): Transform P_bad-violacein constructs into MC1061 - Plate on Spectinomycin

1. Transfrom P_bad-violacein into MC1061 cells:
    - Thaw a 200 uL aliquot of cells on ice
    - Add 50 uL of water to the cells (if greater volume is desired)
    - Add 30 uL of KCM to the cells
    - Dilute P_bad-violacein plasmid from miniprep by 10 (9 ul H2O + 1 ul plasmid DNA)
    - Add 1 uL of dilution to cells and then stir to mix.
    - Let sit on ice for 10 min
    - Heat shock for 90 seconds at 42 degress Celsius 
    - Put back on ice for 1 min
    - Add 100uL of LB and then recover in 37 degree shaker for 1 hour
    - Plate 75 uL on spectinomycin plate and incubate in 37 degree shaker overnight


Day 2 (Wednesday, 4/6): Pick a single colony and grow up overnight to liquid culture

1. Pick a a purple colony
2. Grow a 5 mL LB culture in the 37 degree shaker over night.


Day 3 (Thursday, 4/7): Small scale competent cell prep and P_stress-GFP transformations - Plate on Spectinomycin + Kanamycin

1. Dilute cells by 10 (1.5 mL => 15 mL)
2. Small scale competent prep for the diluted 15 mL of cells:
    - Grow cells in 15 mL LB until cloudy (OD600~0.5) and then put the cells on ice
    - Transfer 4 mL into a centrifuge tube on ice and let cool
    - Centrifuge full speed for 2 minutes and then discard the supernatant
    - Resuspend in 360 uL of TSS solution 
    - Add 40 uL of KCM (total of ~400 uL of competent cells)
3. Transform P_stress-GFP plasmids into newly prepped competent cells:
    - Take ~350 uL of newly prepped competent cells and put it in 35 of 96 well blocks (10 uL per well)
    - Add 50 uL of water to the cells to increase volume 
    - Add 30 uL of KCM to the cells
    - Dilute P_stress-GFP miniprep plasmid by 10 (9ul H2O + 1ul plasmid DNA)
    - Add 1uL of dilution to cells and then stir to mix
    - Let sit on ice for 10 min
    - Heat shock for 90 seconds at 42
    - Put back on ice for 1 min
    - Add 100uL of LB and then recover in 37 degree shaker for 1 hour
    - Plate 75 uL each on 35 spectinomycin and kanamycin wells of 3 agar strip plates
    - Grow upside down at 37 degree overnight in incubator


Day 4 (Friday 4/8): Check plates and store

1. Remove agar strip plates from 37 degree shaker and cover with airtight plastic cover
2. Store in 4 degree fridge 


Day 5 (Monday 4/11): Pick colonies for each P_bad-violacein + P_stress FGP combination

1. Pick and grow 400 uL cell cultures (LB+Spec+Kan) for each P_bad-violacein + P_stress FGP combination in 35 of 96 well blocks 


Day 6 (Tuesday 4/12): Measure GFP/OD in TECAN

1. Dilute cultures 1:100 (396 uL H2O + 4 uL cells for each well)
2. Grow for 2 hours until OD600=0.5
3. Induce with some percent arabinose for optimal growth of P_bad-violacein expressing bacteria (based on data from Team ToxRViol)
4. Measure GFP/OD in TECAN every 30 minutes over 5 hours

Helen Shi 12:39, 5 April 2011 (EDT)

Objective: Transform P_bad-violacein constructs into MC1061 - Plate on Spectinomycin

1. Transformed P_bad-violacein plasmid into MC1061 competent cells and plated on spectinomycin plates
2. Placed plated violacein cells in 37 degree C incubator in Anderson Lab labeled Team Butterfish

Helen Shi 18:25, 6 April 2011 (EDT)

Objective: Pick a single colony and grow up overnight to liquid culture

1. Picked a purple colony and grew a 5 mL LB culture labeled Team Butterfish over night in 37 degree C incubator.

Helen Shi 20:14, 7 April 2011 (EDT)

Objective: Small scale competent cell prep and P_stress-GFP transformations - Plate on Spectinomycin + Kanamycin

1. At 9 AM, diluted the 1.5 mL of purple cells from yesterday's culture in a 15 mL culture
2. Around 10 AM, made 3 spectinomycin and kanamycin agar strips with a total of 35 wells labeled Team Butterfish.
3. At 12:30 PM, the OD600~0.5 so the cells were put on ice for 10 minutes in preparation for small scale competent prep.
4. The cells underwent small scale competent prep, producing ~400uL of competent cells for 35 wells (10 uL per well) with 35 different stress promoter plasmids (1uL).
5. The 35 different stress promoters were then transformed into the P_bad-violacein competent cells on a 96 well plate.

6. 75uL of the transformation from each well was plated on the 3 prepared spectinomycin and kanamycin agar strips (totaling 35 wells), labeled Team Butterfish, and incubated overnight at 37 degrees C.

Helen Shi 21:47, 8 April 2011 (EDT)

Ojective: Check plates and store

1. Plates were checked at 1:45 PM and sealed with plastic cover and labeled Team Butterfish before being stored in 4 degree C deli fridge in the Anderson Lab.
2. Two wells, B5 and F4, failed to yield viable colonies with P_bad-violacein and their respective stress promoters. Perhaps the stress promoters are too sensitive to the toxicity of violacein?

Mary Wang 22:39, 12 April 2011 (EDT)

Objective: Repick P_bad-violacein colony for failed transformations

1. Repicked colonies for failed transformations (F4 and B5) from P_bad-violacein only plate and grew in ~400 uL LB overnight

Mary Wang 22:39, 13 April 2011 (EDT)

Ojective: Small scale competent cell prep and re-transform P_stress-GFP - Plate on Spectinomycin + Kanamycin

1. At 9:30 AM, prepared two competent cell preps by adding 30 uL of culture to 2 different 4 mL of LB. 2. From 9:30am - 12pm, the cultures grew in 37 degree shaker until OD600=0.5. 3. From 12pm - 1pm, made the 2 small scale competent cell preps:

   - Transferred 1mL of culture each for the 2 preps into an eppendorf tube on ice and let cool
   - Centrifuged at full speed for 30 sec and discarded the supernatant for the 2 preps
   - Resuspended in 90uL of TSS solution each for the 2 preps
   - Added 10uL KCM for the 2 preps
   - Added 1uL plasmid DNA each for the 2 different P_stress-GFP parts (F4 - proV/X, B5 - narP)
   - Let sit on ice for 10min, heat shocked 90 sec at 42, iced for a minute, and rescued 1 hr

4. From 2pm - 3pm, plated the 2 transformation on spectinomycin and kanamycin plates.

Mary Wang 22:39, 14 April 2011 (EDT)

Objective: Pick colonies for experimental and control

1. Checked regrown colonies of B5 and F4 parts and found that B5 grew but F4 failed again
2. Picked experimental colonies from agar strips and plate for B5 and grew in 2 mL of LB + KAN + SPEC each in 4 24 well blocks overnight
3. Picked control P_stress-GFP only colonies from frozen stock and grew in 2 mL of LB + KAN + SPEC each in 4 24 well blocks overnight

Mary Wang 22:39, 15 April 2011 (EDT)

Objective: Induce cultures with arabinose and measure GFP expression with TECAN

1. At 8 AM, diluted cultures 1:100 (1980 uL H2O and 20 uL of culture) and grew for 2 hours until OD 0.5 (in 2 mL)
2. At 11 AM, induced with 0.002% arabinose solution and transferred 100 uL from each well into 96 well TECAN plate. 3. From 12-5 PM, placed sample in TECAN and ran TECAN. When we checked TECAN at 5 PM, TECAN had frozen within the first hour so we must remeasure growth curve.

Mary Wang 22:39, 18 April 2011 (EDT)

Objective: Repick and grow cells for TECAN measurements

1. At 12pm, repicked and grew colonies in 400 uL of Kan + Spec + LB per well in a 96 well plate
2. Regrew control colonies in 400 uL of Kan + LB per well
3. Placed into shaker at 2 PM and grew overnight.

Helen Shi 20:45, 19 April 2011 (EDT)

Objective: Induced cultures with arabinose and measured GFP in TECAN

1. At 10 AM, diluted cultures 1:25 (4 uL cells + 296 uL H2O per well).
2. At 2:30 PM, induced with 4 uL of 0.2% arabinose per well (dilute 20% arabinose by 100: 4 uL of 20% arabinose in 396 uL of H2O) 3. At 3:30 PM, transferred 100 uL of induced culture per well into 96 well TECAN plate and started TECAN machine.

Helen Shi 18:26, 20 April 2011 (EDT)

Objective: Obtain data from TECAn

1. Checked TECAN and retrieved OD and GFP expression data from TECAN.

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