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Team:Groningen/Template/MODULE/Notebook/secretion/week6
From 2014.igem.org
September 1 - September 5
Obtaining ssUSP45DspB (BBa_K1365111) as a BioBrick in the pSB1C3 vector and ssUSP45Aiia (BBa_K1365169) as a BioBrick in pSB1C3 vector.
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 gBlock 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 presence of 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
