IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/09/24

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(Protocol)
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#Incubate in ice for 30 minutes, heat shock 42C for 30 seconds and put back in ice for 2 minutes.
#Incubate in ice for 30 minutes, heat shock 42C for 30 seconds and put back in ice for 2 minutes.
#Centrifuge the cells and collect the supernatant.
#Centrifuge the cells and collect the supernatant.
 +
#Dilute the tested cells 10000x (100x then 100x) in LB with antibiotic and corresponding concentration of IPTG.
#Dilute the tested cells 10000x (100x then 100x) in LB with antibiotic and corresponding concentration of IPTG.

Revision as of 20:17, 24 September 2012

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pLac + Colicin E2 Immunity Characterization

We want to characterize the immunity by varying the concentration of IPTG and the ColicinE2 cells.

Protocol

Preparation of the immunity cells

  1. Grow overnight culture of pSG.008 (pLac-Immunity) and pSG.001 (pSB3C5) with antibiotic (Cm).
  2. Centrifuge and resuspend in LB.
  3. Prepare 5 tubes of 10ml LB and put 40uL of pSG.008 cells into 4 tubes and pSG.001 cells in 1 tube (negative control).
  4. Incubate 37C for 30 minutes.
  5. Put IPTG with different concentration in each tube (pSG.008: 100uL, 10uL, 1uL and 0/no IPTG).
  6. Incubate again until the OD reaches 1.0.

Preparation of the colicin supernatant

  1. Grow overnight culture of Colicin E2 cells (pColE2-P9).
  2. Put 40uL of the saturated cells into 10ml fresh LB.
  3. Incubate 37C until the OD reaches 1.0.
  4. Incubate in ice for 30 minutes, heat shock 42C for 30 seconds and put back in ice for 2 minutes.
  5. Centrifuge the cells and collect the supernatant.


  1. Dilute the tested cells 10000x (100x then 100x) in LB with antibiotic and corresponding concentration of IPTG.
  2. Take each 0.5ml and mix with 0.5ml Colicin supernatant (undiluted, diluted 1000x, plain LB with antibiotic and corresponding amount of IPTG) in small 2ml eppendorf tubes.
  3. Incubate for 30 minutes.
  4. Plate 20uL in Cm.

Note: 1ml of cells of OD 1.0 has 1e9 cells, so we expect if all cells survive we will get 1000 colonies at the end.


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