Team:Evry/Notebook/Sensing/PCBs/08-21-2014

Sensor construction bphR2/PbpR1
BBa_J23114 was resuspended with 10 µL sterile water in order to obtain a DNA concentration around 0.2 ng/µl (according to the registry). Solution was transferred into one 1 ml eppendorf tube and stored at -20°C.
Transformations of constitutive promoter BBa_J23114, RFP BBa_E1010 and terminator BBa_B0015 and vector pSB1C3G3 was done on DH5alpha as followed:

1. Remove E. coli competent tubes from -80°C and keep it on ice
2. Add 3 µl of template (here solubilized plasmids from the registry distribution kit) and mix gently
3. Incubate 10 minutes on ice
4. Perform an heat shock 30 seconds at 42°C
5. Incubate 2 minutes on ice
6. Add 2 ml of LB medium and incubate 60 minutes at 37°C with an agitation at 200 rpm
7. Plate 200 µl of pSB1C3G3, BBa_B0015 and E1010 on a chloramphenicol LB agar plate and ampicilline LB agar plate for BBa_J23114
8. Incubate plate overnight at 37°C

We received primers.

pSB1C3 was digested with EcoRI and pstI and a ligation with bphR2 was done before the transformation in DH5a.
50 µL were plated on Cam Lb plate and incubated at 37°C overnight

Aug 21