IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/09/25: Difference between revisions

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==Entry title==
==pLac + Colicin E2 Immunity Characterization==
* Insert content here...
We want to characterize the immunity by varying the concentration of IPTG and the ColicinE2 cells.


===Protocol===
'''Preparation of the immunity cells'''
#Grow overnight culture of pSG.008 (pLac-Immunity) and pSG.001 (pSB3C5) with antibiotic (Cm).
#Centrifuge and resuspend in LB.
#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).
#Incubate 37C for 30 minutes.
#Put IPTG with different concentration in each tube (pSG.008: 100uL, 10uL, 1uL and 0/no IPTG).
#Incubate again until the OD reaches 1.0.
'''Preparation of the colicin supernatant'''
#Grow overnight culture of Colicin E2 cells (pColE2-P9).
#Put 40uL of the saturated cells into 10ml fresh LB.
#Incubate 37C until the OD reaches 1.0.
#Heat shock 42C for 30 seconds and incubate again for 1 hour.
#Centrifuge the cells and collect the supernatant.
#Dilute the tested cells 100x then in LB with antibiotic and corresponding concentration of IPTG.
#Take each 0.5ml and mix with 0.5ml Colicin supernatant (undiluted, diluted 1000x, plain LB with corresponding amount of IPTG) in small 2ml eppendorf tubes.
#Incubate for 30 minutes.
#Dilute 100x in MgSO4 1e-2M
#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.
===Results===
===Results===


Revision as of 03:07, 26 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. Heat shock 42C for 30 seconds and incubate again for 1 hour.
  5. Centrifuge the cells and collect the supernatant.


  1. Dilute the tested cells 100x then 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 corresponding amount of IPTG) in small 2ml eppendorf tubes.
  3. Incubate for 30 minutes.
  4. Dilute 100x in MgSO4 1e-2M
  5. 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.

Results

100uM IPTG 10uM IPTG 1uM IPTG 0 IPTG (-) control
1x Colicin 25 37 45 78 101
1000x Colicin 89 67 149 200 152
No Colicin 80 77 41 386 7


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