SBB11Ntbk-Team Butterfish

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. Helen, Justin, and Chia transform 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. Chia comes in the morning to pick a purple colony and grows 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, Chia diluted the 1.5 mL of purple cells from yesterday's culture in a 15 mL culture
2. Around 10 AM, Justin makes 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 are put on ice for 10 minutes in preparation for small scale competent prep.
4. The cells undergo 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 is plated on the 3 spectinomycin and kanamycin agar strips (totaling 35 wells) prepared by Justin, 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. Repick colony for failed transformations (F4 and B5) from P_bad-violacein only plate and grow 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, Chia prepared two competent cell preps by adding 30 uL of culture to 2 different 4 mL of LB. 2. From 9:30am - 12pm, the culture grew in 37 degree shaker until OD600=0.5. 3. From 12pm - 1pm, Su and Chia Hung 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
   - Add 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, Justin 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. Pick experimental colonies from agar strips and plate for B5 and grow in 2 mL of LB + KAN + SPEC each in 4 24 well blocks pick
3. Pick control P_stress-GFP only colonies from frozen stock and grow in 2 mL of LB + KAN + SPEC each in 4 24 well blocks pick

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

8am- 10am Su Dilute cultures 1-25. grow for 2 hours until OD 0.5 (in 2mL )

10am CH will induce with 20uL arabinose solution. mix. take 100 uL from each well and put into 96 well plates. place in tecan.

11am Justin picks up CH slack if we don't grow if to .5 OD in time

12-5pm Sample in Tecan- TECAN froze so repeat growth curve.

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

12pm In a 96 well block, I labeled one side in red tape, the control and the other side in yellow tape. A1 is in the top left for both- rotate the channel in order to obtain this configuration. A-H is along width, 1-5 column is along length. Control: Kan + LB (400uL) in each well using a multichannel pipet Colonies: Kan+ Spec + LB in each well (400uL) using a multichannel pipet Pick using a flamed toothpick the controls and the colonies into the matching well. Placed into shaker (~2pm).

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

10:00 AM Dilute 1:100 (4 uL cells + 296 uL H2O per well)

2:30 PM Induce with 4 uL of 0.2% arabinose per well (Dilute 20% arabinose by 100: 4 uL of 20% arabinose in 396 H2O)

3:30 PM Transfer 100 uL of induced culture per well into TECAN plate and start TECAN machine