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

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

Repick the failed colonies from original violacin and throw toothpick in LB to grow overnight.

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

-Transform the corresponding stress promoters into the two failed parts (F3 and B5). -Turn the overnight cultures to competent cells through competent cell prep -grow to 0.5 OD -heat shock stressPromoter-gfp into the competent cells.

Time BreakDown

    TOTAL ESTIMATED TIME: 6 hours. 9:30am -3:30pm
  

9:30am - 12pm Chia Hung will grow diluted culture to .5 OD. Follow protocol exactly except use all 4 mL of shit.(30 uL into 4 mL and shake) 12pm - 1pm. Su + Chia Hung

Repeat twice. each step. 1) Transfer 1mL into an eppendorf tube on ice, let cool 2) Centrifuge full speed for 30 sec, toss out supernatant 3) Resuspend in 90uL of TSS solution 4) Add 10uL KCM

 Step 4.5:  do the negative control before adding the DNA

5) Add 1uL plasmid DNA 6) Let sit on ice for 10min, heat shock 90 sec at 42, ice for a minute, rescue 1 hr, then incubate and/or plate

2pm - 3pm Justin PLATE

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

Failed part B5 grew however failed part F3 did not. Put 2mL of LB+ KAN + SPEC in 4 24 well block. Pick colonies for control. Pick colonies for colonies.

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.