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
- 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
|
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
|
|