Team:Groningen/Labwork/12 August 2013



did a miniprep of CFP 1,2 and 4 and Pdes CheY 1 and 2 and a gell purification of Munich EcoRI*PstI


Made -80 stock of strains OI3017 OI3059 OI2653 OI3135.
Made a setup to test motility, using 0.4% agar test-tubes with different obstacles in them.
4 sets of 3 tubes are stabbed with a toothpick, one negative, one with Bacillus subtillus gfp backbone, and one OI3017.
The OI3017 should not be motile the Bacillus subtillus gfp should be motile and the negative control should be negative.
The normal test was done in duplo (due to a failed abstacle preparation). the other test are 3 ml agar on top of the normal level. And a cone (salvaged from a 5 ml pipet) seperating the strain from air.
Tubes are to grow overnight in a 37C incubator. Except one of the duplo series which was put in the shaker to see the difference.


Inocculated GFP 1&2 YFP 1&2 1:50 in 20 ml of LB.
At OD600=~0,4 took sample T0 and added IPTG to 1mM.
Grew for 2 hours and checked under the microscope.
YFP1 did not give any signal.
GFP1 gave a weak signal for T0&T2

Need to do a better setup with B. subtilis 168 wt as negative control.
so, inoculated GFP 1&2 and B.sub168 wt.

GFP was taken from the BioBrick system () and is not optimized for B. subtilis. eYFP BBa_E0030 didn't had an RBS and this may cause no translation of the protein.