Team:Groningen/Labwork/12 September 2013

Claudio

The plates pSB1C3-S1-S5-S5 and pSB1C3-S2-S5-S5 showed thousands of colonies.
Colony PCR was performed on 4 colonies per plate using VF2 and VR primers (annealing temperature 58°C).
The samples were checked on agarose gel 0.8%.
Colony C from pSB1C3-S1-S5-S5 and colony B from pSB1C3-S2-S5-S5 were positive candidates and were inoculated in LB + Cm.

PCR was performed using S11 as template, S16-F and S16-R as primers (annealing temperature 63°C) and NEB High Fidelity TAQ.
The PCR product was purified.
pSB1C3-S16-S3 and pSB1C3-S16-S9 were digested with SpeI and PstI.
S11 was digested with XbaI and PstI.
S5 (previously digested with XbaI and PstI) and S11 were ligated into pSB1C3-S16-S3 and pSB1C3-S16-S9, respectively.
The ligation products were transformed in E. coli DH5α and plated on LB + Cm.

The ligation products from yesterday, BBa_K1085014-S1-S5-S13 and BBa_K1085014-S2-S5-S14 were transformed in E. coli DH5α and plated on LB + Amp.

Mirjam

Did a ligation of the silk plus signal sequences (made by Sander) to the registration backbone.
Saw colonies on the plates of complete transformation of the ΔCheY mutant to our B. subtilis 168 strain.

Inne

Prepared the samples of biobrick BBa_k1085012 and BBa_k1085010 for sequencing. 4 samples were prepared.

Biobrick	Primer
BBa_k1085012 5 uL	VF2 5 uL
BBa_k1085012 5 uL	VR 5 uL
BBa_1085010	VF2 5 u/L
BBa_1085010	VR 5 u/L

Sebas

Checked B. subtilis with GFPdsm and GFP0840 and WT in the amyE locus, under the microscope.
Made phase constrast pictures (white light, 0,4s exposure 32%) and with GFP filter (0.5s exposure, 50%.

Induced strains showed significantly more fluorescence than WT, see: BBa_K1085014.

Sequenced RFP strain form 11-09 was not the right one. So inoculated 16 other colonies still stored in the fridge.