Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 22nd August.html

Trojan Horse

22^nd August

Results :

All the transformation worked

Launch cultures (to do experiment)

• mGZ1 F+ -Cola

• MGZ1 F+ litmus + Helper

Launch overnigh culture to do glycerols.

exploring the parameters in play in the phage infectiveness of our new phagemid construct

-diluting 100x O/N of MGZ1, F+

-mix with 1/10 vol of supernatant containing phagemids with the cells with the following combination of parameters:

OD600 : 0.3 0.6 0.9

Plate at different time (min) after first contact with the supernatant: 2, 30, 60

Silencing experiments

- Launch O/N culture of producing phages cells ; centrifuge ; take supernatant and filter it.

-diluting 100x O/N of MGZ1, F+ -pCola in LB-Kan

- At OD 0,7 : 4 tubes : Ctrl, P1, P2 ,P3. Put 1 mL of cells in each tube. Centrifuge. Take out supernatant, resuspend in 1mL of LB. Add in tubes respectively : nothing, 100uL phages of Miniprep 1, Miniprep 2, miniprep 3.

- Incubate 45 minutes at 37°C

- Serial Dilute every tube until 10-5. Plate for each tube 100ul of 10-5 dilutions on Kan and LB and 100 ul of 10-2 and 10-3 dilution on Kan.