Team:Groningen/Template/MODULE/Notebook/secretion/week4

The Biobricks pLAS, P2, GFP, RBS and Double terminator were assembled using three-A assembly, the constructs were ligated, and transformed to E. coli DH5α

The transformations were plated on LB agar with kanamycin

All the colourless colonies were plated to fresh plates again, and grown overnight

The construct was checked by colony PCR, ending up with the following constructs: Ps with RBS, pLas with RBS, and the gene coding for GFP with double terminator

Another colony PCR was performed with Phusion polymerase this time, in order to produce a more clean product for a second assembly

The primers and the synthetic gene ssUSP45DspB (BBa_K1365111) arrived

The mrfp insert was cut out of pSB1C3, run on gel, and the linearized vector was purified out of the gel

ssUSP45DspB was ligated with pSB1C3, and transformed to E. coli DH5α

Few colonies were visible after growing the transformation mixture of LB agar plates with chloramphenicol

After digestion, the constructs of the few colonies were checked on gel, only one colony showed the right size for the insert