Team:Groningen/Template/MODULE/Notebook/Secretion

Performing three transformation to E. coli, one with promoter P2 (BBa_I746104), one with lasI (BBa_K081009), and one with aiiA (BBa_C0060)

The tranformated E. coli was divided in several concentrations over LB agar plates with Chloramphenicol

Three glycerol stocks of E. coli DH5α containing the RBS (BBa_ B0034), the double terminator (BBa_ B0015) and the promoter LasR (BBa_ R0079), were inoculated in LB medium, and grown overnight

The plasmids were isolated out of the overnight cultures, and checked on gel

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

An overnight culture of E. coli DH5α with the P2 with RBS construct, pLas with RBS construct, and the gene coding for GFP with the double terminator construct was made

The constructs P2 with RBS, pLas with RBS, the gene coding for GFP with the double terminator and ssUSP45DspB were checked on gel, only P2 with RBS and pLas with RBS had the correct size

The same experiment with the ssUSP45DspB was performed again, but again no positive results were obtained

PCR was performed with the synthetic ssUSP45DspB gene, resulting in ssUSP45 and ssUSP45DspB without the His-tag

Two PCR reactions directly on the products were performed

The products were ligated with pSB1C3, and transformed to E. coli DH5α

Another PCR was done with the amplificated PCR product of ssUSPdspB, one with the signal sequence of USP45, and one with the ssUSP45DspB without His-tag

These products were also ligated with pSB1C3, and transformed to E. coli

Afterwards, Gibson assembly had been done with modified Aiia and the signal sequence of USP45 making ssUSP45aiiA, to enhance the chances of successfully ligating the Gibson product into pSB1C3, PCR was done on the final Gibson product

The PCR product was checked on gel, giving positive results for the ssUSP45aiiA, then it was ligated with pSB1C3, and transformed to E. coli

Several colonies were visible on the plated transformation products

Colony PCR was done on them with the regular pSB1C3 test primers, which showed no results

The colonies were grown overnight, and the plasmids were isolated in the next week