Team:Paris Bettencourt/Notebook/Trojan Horse/Wednesday 31st July.html

Trojan Horse

31^th July

Vincent and Aude

Conjugation MGZ1, F+ & MG1655:

Protocol :

1. From O/N cultures Dilute both strains 1/100 , in LB

2. Wait for OD to reach O,2

3. Prepare 4 tubes (in BD tubes) :

• Tube 1 = 0,5mL LB with Strain 1 (Here MGZ1) + O,5mL LB = control

• Tube 2 = 0,5mL LB with Strain 2 (here sT004) + 0,5mL LB = control

• Tube 3 and 4= 0,5mL LB with Strain 1 (Here MGZ1) + 0,5mL LB with Strain 2 (here sT004

4. Incubate 2 hours at 37°C

5. Plate 50ul for controls, 50uL, 20ul, 10ul for mixed tubes on LB antiobiotics (here tet and spec)

6. Incubate overnight at 37°C

Vincent

Infectiveness characterization experiments

-the night before: prepare O/N of F+ cells, prepare O/N of phagemid producing cells (in this case : sT007 )

-centrifugate phagemid producing cells

-filter the supernatant 0.45um, stock it at 4°C (it contains the phages)

-dilute 200x MGZ1, F+ from O/N

-wait until OD600 = 0.7

-immediately mix MGZ1 and the supernatant in different proportions

here we planned to try 1/100 (vol supernatant/vol cells), 1/1000 and 1/10000

Unfortunately the supernatant was dropped on the bench.