Team:Groningen/Labwork/2 August 2013

Mirjam

Plates with eYFP plated on LB agar with IPTG and amp look more yellow when the construct is transformed correctly. To be certain about this the samples are inoculated in LB medium with IPTG (0M, 0.1mM and 1mM) and amp.
Plates with the transformants to B. subtilis did not show any significant results.
Picked colonies of the Pdes/CheY plate and inoculated in LB medium with ampicillin.
Made a new ligation of Pdes and CheY. Transformation to E. coli.

Claudio

The plates prepared yesterday show colonies. Unfortunately the colonies are red which means the ligation didn't work.

The strains inoculated by Inne grew. -80°C stock of AKP4 (Δdes::kn::cm) and OIB055 (ΔcheY::cm) was made and stored in the box labeled iGEM 2013.

BBa_K823023 + lacI, BBa_E0840 and BBa_E0240 are digested with EcoRI and PstI. The samples are purified using the PCR purification kit. To the tube containing the purified vector 1µl of SpeI and XbaI is added and the tube is incubated 1h at 37°C. Afterwards the enzymes (SpeI and XbaI) are heat inactivated. BBa_K823023 + lacI is ligated to BBa_E0840 and BBa_E0240 (3:1 insert:vector ratio). The ligation products are transformed to E. Coli DH5α and the competent cells are plated on LB + Amp.

2x SG plates are poured and 2µl of the Bac. Sub. 168 liquid culture grown overnight is spotted on agar. The plates are dried and then incubated at 30°C as long as Bac. Sub. biofilm will form.

Sander

did miniprep of 4 mKate 4 BBa_E0240 and 4 BBa_E0840 inoculations, then did a restriction digest on them with EcoRI and PstI. ran the digest over a 0.8 % agarose gel 90V 40 min.

The digestion check shows one positive candidate (GFP0840 C) which shows one band at 7529 bps (backbone) and 919 bps (RBS-GFP). File:File-Dig check 2 2013-08-02 17hr 48min.JPG