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): Mon (4/11): Gary and Averee come in and perform large scale transformation: All stress promoters:GFP + one Con:GFP + 1 (-) control (36 transformations)
 * Placed plated toxR cells in 37 degree C incubator labeled with M.A.N.G, date and time.
 * Marcus went in at 15:10 and picked colonies.
 * Labeled tube with red tape (BE 140L, Team M.A.N.G, date+time), placed in Warm Room (448A Stanley) in rack on the shaker closest to entry door on left side of room.
 * Stored our plated colonies in class room lab's glass door refridgerator on 2nd rack from top, Labeled accordingly.
 * See Picking Colonies for in-depth procedures as suggested by S. Boyarskiy.
 * 1) Gary comes in at 9:30am. He dilutes the saturated 3ml culture into a flask full of 27ml LB. After 2 hours, performs small scale competence protocol. After resuspending in TSS (90ul x the number of starting ml of cells) freeze the cells in -80.
 * 2) Nikit and Marcus prepares 6 strips

The negative control was plated onto its own round S/K plate. Tuesday 4/12 Nikit picks colonies and we all plan our next move during class time.


 * for Tuesday (4/5)
 * 200&micro;L of MC1061 competent cells
 * 1 toxR miniprep
 * spec plate

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 1800 uL (200 uL ea) aliquot of MC1061/toxR competent cells on ice
 * 2) Add 450 uL (50 uL ea)  of water to the cells (if greater volume is desired)
 * 3) Add 270 uL (30 uL ea)  of KCM to the cells
 * 4) Put your miniprep mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
 * 5) Add 30 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 selective antibiotics on petri strips, let incubate at 37 degrees overnight