Team:Groningen/Labwork/11 September 2013

Sebas

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.

Claudio

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.

Sander

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)