SBB11Ntbk-Team ToxRViol

15:07, 21 April 2011 (EDT)

today we came into lab and analyzed our data from the Tecan

15:20, 12 April 2011 (EDT)

Induce:
--make 250uL 20% arabinose solution:
add 50mg Arabinose to ~.25g LB+Spec
--make 2 mL .2% Arabinose solution:
take 20uL of 20% arabinose solution, add to 1880 uL Lb+Spec
--make 2 mL .02% Arabinose solution:
take 200uL of .2% arabinose solution, add to 1800 uL Lb+Spec
6.9) make stock solutions of arabinose. Using LB+SPEC
%Arabin uL(20% or .2% or .02%) to add to 10mL LB+Spec
0.0 0.0
1.931e-06 .9655
5.517e-06 2.7585
1.576e-05 7.88
4.504e-05 2.252
0.0001287 6.435
0.0003677 18.385
0.00105 52.5
0.003 1.500
0.00858 4.290
0.0245 12.250
0.07 35.0
0.2 100.0

17:34, 11 April 2011 (EDT)

Pick colonies from plates in 4 C fridge one of ToxR, Violacein, and control in 3mL LB+Spec media

13:34, 5 April 2011 (EDT)

1.) Transform (heatshock) nontoxic gene plasmids into MC1061, plate +Spec
2.) Transform(heatshock) toxic gene plasmids into MC1061,plate +Spec

Procedure

 1. Thaw a 200 uL aliquot of cells on ice

 2. Add 50 uL of water to the cells (if greater volume is desired)

 3. Add 30 uL of KCM to the cells 

 4. Put your ligation 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 ligation, 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, let incubate at 37 degrees overnight

Protocol as of 4-6-2010

List of Materials: Bac Culture of: pBCA1256-BCA1144 1766-BCA1144 pBCA9523-BCA1144 ToxR- pBca1256-bdc004-> Plasmid for ToxR named bdc022 Violacein- pBca9523-jtk2914 +Spec LB (200mL) Arabinose x grams MC1061 4 Spec Plates

Goal: Evaluate toxicity of ToxR and Violacein(spec)

0.) Do the following procedure once per Toxic gene: ToxR(Antibiotic = Spectinomycin, plasmid = pBCA1256-BCA1144(RFP) Violacein(Antibitoic = Spectinomycin, plasmid = pBCA9523-BCA1144(RFP))

CONTROL: 0.) Prepare a plasmid without the toxic gene- use RFP instead

Overnight: 1.) Transform (heatshock) nontoxic gene plasmids into MC1061, plate +Spec, pick colony, and grow in 2mL liquid media in a 10mL tube in LB + Spec overnight at 37C {O/N Control}

2.) Transform(heatshock) toxic gene plasmids into MC1061,plate +Spec, pick colony, grow in 2mL liquid in a 10mL culture tube in LB +Spec overnight at 37C {O/N Toxic}

1. Thaw a 200 uL aliquot of competent cells on ice

2. Add 50 uL of water to the cells (if greater volume is desired)
3. Add 30 uL of KCM to the cells- did not add KCM, volume cells too small
4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)- did not dilute miniprep product
5. Add 70 uL of the cell cocktail to the ligation, stir to mix- added 2uL miniprep plasmid/10uL diluted & 50uL cells
6. Let sit on ice for 10 min- waited 7 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- used LB media

10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight- plate 50uL on Spec plates

Prep: 3.) add 2mL LB + Spec to 10x 10mL test tubes 4.) add 40 uL of O/N Control MC1061 to culture #1a-5a 5.) add 40 uL of O/N Toxic MC1061 to cultures #1b - #5b 6.) grow until OD(600nm) of the cultures is .4 - .6 , on a shaker at 37C

CHECK!!!! IF CUVETTES WTO MEASURE MID LOG PHASEHAVE ENOUGH BACTERIA

Induce: --make 250uL 20% arabinose solution: add 50mg Arabinose to ~.25g LB+Spec --make 2 mL .2% Arabinose solution: take 20uL of 20% arabinose solution, add to 1880 uL Lb+Spec --make 2 mL .02% Arabinose solution: take 200uL of .2% arabinose solution, add to 1800 uL Lb+Spec 6.9) make stock solutions of arabinose. Using LB+SPEC %Arabin uL(20% or .2% or .02%) to add to 10mL LB+Spec 0.0 0.0 1.931e-06 .9655 5.517e-06 2.7585 1.576e-05 7.88 4.504e-05 2.252 0.0001287 6.435 0.0003677 18.385 0.00105 52.5 0.003 1.500 0.00858 4.290 0.0245 12.250 0.07 35.0 0.2 100.0

7.) Add Arabinose to a final concentration of: tube# 1t 0.0 2t 1.931e-06 3t 5.517e-06 4t 1.576e-05 5t 4.504e-05 6t 0.0001287 7t 0.0003677 8t 0.00105 9t 0.003 10t 0.00858 11t 0.0245 12t 0.07 13t 0.2

→ http://www.pnas.org/content/99/11/7373.full- Arabinose Concentrations

8.) Post induction, Load 100uL from each test tube onto a plate to go into a Tecan plate reader- Do this twice per test tube so that all samples are duplicates.

a.) Setup Tecan to measure OD(600nm) of the each sample in the plate every 15-30 minutes overnight

9.) Copy results from plate reader to powerpoint 10.) average data from duplicate wells 11.) setup a 3D graph time v. [arabinose] v. OD (or alternatively, 5 2D graphs OD v. time) a.) if the parts are toxic, we expect slower growth and a lower final OD