Team:Groningen/Labwork/11 September 2013



Checked sequenced plasmids. BBa_K1085014+GFPdsm and BBa_K1085014+GFP0840 had both the promoter orientated in the right direction.
There were no mutations.

BBa_K1085014+P-RFP, only one primer bound in the wrong direction, implying that the promoter is inserted in the wrong direction. The other primer didn't bound at all.

checked colonies stabbed on LB/starch/cm plates, with lugols solution. No halo's were visible implying that the B. subtilis colonies have an insert in the amyE locus.

New transformation of BBa_K1085014+GFPdsm had ~100 colonies (Negative control was clear)
Stabbed 3 colonies on LB/Starch/cm plate and streaked on LB/cm plate.
Inoculated 3ml LB with GFPdsm col 1.
Sent RFP col1 for sequencing:

153DZAB027 RFP1 Fw 153DZAB028 RFP1 Rv

Inoculated 3ml LB/amp with RFP colonies 5-8.

Inoculated 3ml LB with B.sub BBa_K1085014+GFP0840 and B sub WT.


S5 was ligated into pSB1C3-S1-S5 and pSB1C3-S2-S5.
The ligation product was transformed into E. coli DH5α and plated on LB + Cm.

The plates from yesterday (pSB1C3-S16-S3 and pSB1C3-S16-S9) showed around one hundred colonies.
ColonyPCR was performed on 4 colonies per plate using VF2 and VR primers (annealing temperature 58°C).
The PCR samples were checked on agarose gel 0.8%.

Colony C from pSB1C3-S16-S3 and colonies C and D from pSB1C3-S16-S9 were positive candidates. Indeed the gel showed bands at the expected height (1219 bps).
Colony C from pSB1C3-S16-S3 and colonies C from pSB1C3-S16-S9 were inoculated in LB + Cm (Sander).

BBa_K1085014-GFP0840, pSB1C3-S1-S5-S13 and pSB1C3-S2-S5-S14 were digested with EcoRI and PstI.
The digestion product were purified from gel.
Both S1-S5-S13 and S2-S5-S14 were ligated into BBa_K1085014. The ligation reaction was incubated over night.


made - 80 and a miniprep of the S1-S5-S13 and S2-S5-S14.
the plasmids acquired with the miniprep were send to get sequenced.
did a ligation reaction of F and S3 (200ng 1:1), M and S9 (150 ng 1:1), L and S3 and L and S9 (70 ng 1:1)


Run a gel for the overnight PCR of the ΔCheY and ΔDes strain with as control the primers for the ΔDes mutant on the wild type B. subtilis strain. For the CheY mutant no bands were present at the gel, or the Des mutant, there were more bands than expected and at a different size. The control showed a smear.
Did a restriction reaction on the signal sequences LytB, EstA, MotB and FliZ with EcoRI and BamHI. Heat inactivated the reaction.
Did a restriction reaction on BBa_J04450 with EcoRI and PstI. Heat inactivated the reaction.