Paris/August 29
From 2007.igem.org
Att Biobricks
We got clones. We spread in 5 mL of LB (Kanamycine) culture one of them, for doing minipreps and glycerol stocks.
Expression test of DapA promotor by differential DAP repression
The principe is to get W121 cells rid of internal DAP that coul remain after O/N culture (as show the growth kinetic graphs). We decide then to dilute 1000X the original culture in 10 mL of LB DAPless and make growing bacteria on their stocks during 3-4 hours at 37°C under agitation.
For differential repression, we'll used four DAP concentrations and a control :
- 0 µM
- 10 µM
- 50 µM
- 100 µM
- 200 µM
We make growing bacteria within their respecting broth in 2 mL during 1/2h at 37°C under agitation.
Then we can prepare the slides with gel for microscopy.
We concentrate the 2mL of culture by centrifugation
- Remark : after centrifugation we haven't got any pellet...
We decide to try anyway but we had lost every cells except some. We'll try again tomorrow with a 100X dillution instead of 1000X, and we'll use a 50mL broth for differential represion. We should get more cells.
Starting a new O/N culture of W121 [DapaAp>>>mRFP]
