SBB11Ntbk-Team Butterfish

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

Goal: Evaluate induction of stress by toxic genes in the promoter set for violacein In essence, put Pbad-violacein in the cell, put a stress promoter-GFP in the cell, add arabinose until it starts getting toxic, measure the GFP on a plate reader (the Tecan).

General Procedure Timeline:

Day 1 [Tuesday 4-5]: Heat shock P_bad.violacein constructs into MC1061 - Plate on Spectinomycin  / we also need to begin setting up for control experiment - ask Sergey for materials

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.
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
For Miniprep product (P_bad.violacein plasmid), dilute by 10, (9ul H2O + 1ul plasmid DNA) then use 1uL of dilution to add to cells. Stir to mix.
Let sit on ice for 10 min
Heat shock for 90 seconds at 42 (longer incubation may work better)
Put back on ice for 1 min
Add 100uL of LB, let shake in the 37 degree incubator for 1 hour
Plate 70+ uL on Spectinomycin plate, let incubate at 37 degrees overnight


Day 2 [Wednesday 4-6]: Pick a single colony and grow up overnight to liquid culture

	1. Select a purple colony
	3. Grow 5 mL LB culture over night.

Day 3 [Thursday 4-7]: Small scale competent cell prep - transform P_stress plasmids - plate on Spectinomycin + Kanamycin

Dilute cells in morning [1.5 mL => 15 mL?]

From openwetware:
SMALL SCALE - needs to be set up for ~15 mL cells
Grow cells in ~4 mL LB [15mL?] until cloudy (OD600=0.5)
Put on ice
Transfer 1mL [15mL] into an eppendorf tube [3 different 5mL centrifuge tubes?] on ice, let cool
Centrifuge full speed for 30 sec [2 min.?], toss out supernatant
Resuspend in 90uL of TSS solution [90 ul TSS/1mL pelleted cells?]
Add 10uL KCM [10ul KCM/1mL pelleted cells?]


[at this point, we would probably start following our protocol below, where did we get 300 ul from?]

//Step 6.5:  do the negative control before adding the DNA

7. Add 1uL plasmid DNA
8. Let sit on ice for 10min, heat shock 90 sec at 42, ice for a minute,
rescue 1 hr, then incubate and/or plate on Kan + Spec//

From our protocol:
Part2 - Promoter-GFP Transformation 
	-take (300 uL) our competent cells and put it in 35 of 96 well block
Add 50 uL of water to the cells (if greater volume is desired)
Add 30 uL of KCM to the cells
For Miniprep product (P_stress.ffGFP plasmid + Pbad.VioABCDE), dilute by 10 (9ul H2O + 1ul plasmid DNA) then use 1uL of dilution to add to cells. Stir to mix.
Let sit on ice for 10 min
Heat shock for 90 seconds at 42 (longer incubation may work better)
Put back on ice for 1 min
Add 100uL of LB, let shake in the 37 degree incubator for 1 hour
Plate 70+ uL on plate with both spectinomycin and kanamycin, let incubate at 37 degrees overnight [plate on strip plates]



Day 4 [Friday 4-8]: Check plates and place in 4 deg fridge.

Day 5 [Monday 4-11]: Pick colonies into liquid culture 24 well blocks [LB+Spec+Kan]
Day 6 [Tuesday 4-12]: Dilute cultures 1:100 [8AM], take time points every 30 min., grow to OD 0.5 [~10-11 AM], induce with arabinose, grow for 3 hours in 37 deg shaker, measure GFP/OD in TECAN [~2PM]

Wednesday/Thursday -> can repeat on other colonies, if the assay doesn’t work