SBB11Ntbk-M.A.N.G.

BE 140L Spring 2011 Team MANG
Marcus Macaulay
Averee Chang
Nikit Patel
Gary Dixon

Goal: Evaluate induction of stress by toxic genes in the promoter set for toxR. In essence, put Pbad-toxR 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).

'Schedule;
Tues (4/5): Marcus comes in at 9am transforms ToxR plasmid into MC1061 compitent cells. Plate on SPEC.
Wed (4/6): Nikit comes in at 9pm pick a colony and culture it in 3ml LB (tell GSI). .
Thur (4/7):

1. Gary comes in at 3:30pm. He dilutes the saturated 3ml culture into a flask full of 27ml LB. Measures OD every 45min.
2. Averee comes in at 5pm and takes over. When OD=.5, she performs small scale competence protocol. After resuspending in TSS (90ul x the number of starting ml of cells) freeze the cells in -80.
3. Nikit and Marcus prepares 6 strips

Fri (4/7): Everybody comes in at class time and performs large scale transformation:

1. All stress promoters:GFP and the one Con:GFP into ToxR cells (36 wells)
2. All stress promoters:GFP into non ToxR cells (original) (35 wells)
3. Constitutive promoter:GFP into non ToxR cells (original) (1 wells)

Things we have to tell/need from GSI:

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for Tuesday (4/5)

• 200µL of MC1061 competent cells
• 1 toxR miniprep
• spec plate

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Procedure:

Transformation #1 (Marcus) - we need 1750 uL TSS mixture

1. Thaw a 200 uL aliquot of MC1061 competent cells on ice
2. Add 50uL of water
3. Add 30 uL of KCM to the cells
4. Put your toxR miniprep mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
5. Add 70 uL of the cell cocktail to the miniprep,, stir to mix
6. Let sit on ice for 10 min
7. Heat shock for 90 seconds at 42 (longer incubation may work better)
8. Put back on ice for 1 min
9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on SPEC plate, let incubate at 37 degrees overnight

Preparing Competent Cells:

1. Pick colony from Spec plate and grow in 3mL liquid media overnight (14-16 hrs.)
2. Dilute 10% in fresh media. (Add 3mL culture in 27mL media flask) = 30mL solution
3. Take OD measurements every half hour to make sure cells are in mid-log phase. ~ 2 hours.
4. Do competent cell prep by adding 90uL of TSS for every mL of media left in the tube.
5. Freeze flask?

Transformation #2

1. Thaw a 200 uL aliquot of MC1061 competent cells on ice
2. Add 30 uL of KCM to the cells
3. Put your toxR miniprep mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
4. Add 70 uL of the cell cocktail to the miniprep, stir to mix
5. Let sit on ice for 10 min
6. Heat shock for 90 seconds at 42 (longer incubation may work better)
7. Put back on ice for 1 min
8. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
9. Plate 70+ uL on selective antibiotics on petri strips, let incubate at 37 degrees overnight