Team:Groningen/Labwork/14 August 2013



Ligation reaction of ΔCheY and ΔDes (with help of Chaline)
Transformation to B. subtilis ΔDes transformed in ΔCheY and ΔCheY into ΔDes.
Inoculation of Pdes-CheY-GFP in LB medium with ampicillin (although strange colonies grew on the negative control plate). The colonies didn't grew in liquid LB medium.


made 3 inoculations of the cheY knockout and 3 inoculations of wild type bacillus in 0,4% agar with LB medium to test motility.


Did a restriction on CheY down and Des up with EcoRI and heat inactivated the samples.
Run the samples over gel and the bands are visible.
Measured the concentration of CheY down and Des up.


Made an inoculation of E.coli from Utah, first in a Erlenmeyer with 200 ml LB medium, 500 uL Cm (5mg/mL) and 2 mL of previously made culture.
Then pipetted 3mL of this into a test-tube to grow overnight at 37C.
The motility assay didn't provide the results we were looking for. The Negative control of tubes with pipet tips was contaminated, the other tubes did show colonies in small dots in the agar of the test tubes. This is probably from bacteria floating up when the agar was poured.


Grew B subtilis hyperspank::GFP 1 and induced at OD=0,3 with 1mM and 10mM IPTG.
No significant difference between wt and induced. The GFP or promoter does not work. Because the promoter does
work in a non-biobrick compatible backbone probably the GFP doesn't work in B. subtilis.

Inocculated E.coli harboring a plasmid with a different GFP that worked in B. subtilis before.

Digested MS2(MotB-strep-silk)with XbaI and PstI and ligated it into the hyperspank plasmid.
Transformed it to E. Coli.