IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/09/23

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Current revision (21:24, 24 September 2012) (view source)
 
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#Grow overnight culture pSG.008 (pLac-Immunity) and pSG.001 (pSB3C5) with antibiotic (Cm).
#Grow overnight culture pSG.008 (pLac-Immunity) and pSG.001 (pSB3C5) with antibiotic (Cm).
#Centrifuge and resuspend in LB.
#Centrifuge and resuspend in LB.
-
#Prepare 5 tubes of 10ml LB and put 10uL of pSG.008 cells into 4 tubes and pSG.001 cells in 1 tube.
+
#Prepare 5 tubes of 10ml LB and put 40uL of pSG.008 cells into 4 tubes and pSG.001 cells in 1 tube.
#Incubate 37C until the OD reaches 0.2
#Incubate 37C until the OD reaches 0.2
#Put IPTG with different concentration in each tube (pSG.008: 0.1mM, 0.05mM, 0.025mM and 0/no IPTG).
#Put IPTG with different concentration in each tube (pSG.008: 0.1mM, 0.05mM, 0.025mM and 0/no IPTG).

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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

  1. Grow overnight culture 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.
  4. Incubate 37C until the OD reaches 0.2
  5. Put IPTG with different concentration in each tube (pSG.008: 0.1mM, 0.05mM, 0.025mM and 0/no IPTG).
  6. Incubate again until the OD reaches 1.0
  7. Take each 0.5ml and mix with 0.5ml Colicin cells (saturated, saturated diluted 1000x, plain LB with corresponding amount of IPTG) in small 2ml eppendorf tubes.
  8. Incubate for 30 minutes.
  9. Dilute 10000x (100x then 100x) in MgSO4 e-2M.
  10. Plate 20uL in Cm to kill the Colicin cells and leave the immune cells.

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

The table shows the number of colonies survive in each plate IPTG x Colicin.

Image:SG_exp230912.png

In this experiment we had some weird things which we try to explain

  • The plates have more cells when they have more Colicin; the Colicin maybe take the Immunity/Cm-resistance plasmid
  • Less cells on the plate without Colicin; the cells may have died because they loose the Immunity/Cm-resistance plasmid during the incubation without antibiotic (which we did to avoid killing the Colicin cells)

Solution for the next experiment: instead of mixing cells with Colicin cells, we will mix them with the supernatant of centrifuged Colicin cells (only the ColE2 protein). Therefore we can put the antibiotic during the whole experiment to avoid plasmid loss.

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