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

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11 - 15 of August
 
goal: assemble the promotors with RBS and GFP (BBa_E0040) with double terminator.
 
Biobricks pLAS, P2, GFP, RBS and Double terminator were assembled accordingly to their place in the construct with 3A assembly, ligated, chemically transformed and inoculated on kanamycine agar plates.
 
The O/N grown colorless clones were picked and grown on their own individual plate. Afterwards, colony PCR on all the colonies showed a positive result for assembled: P2+RBS, pLAS+RBS, GFP+Dterm.
 
After the colony PCR, another one was done with phusion DNA polymerase, so that the product could be used for a second assembly with (P2+RBS)+(GFP+Dterm) and (pLAS+RBS)+(GFP+Dterm).
 
Corresponging in only a few clones which gave negative results after colony PCR. Therefore, another assembly with the constructs should be done.
 
Primers came in, preparing them accordingly.
 
The synthetic gene ssUSP45DspB, which arrived from IDT, was prepared according to the protocol made by IDT.
 
1µl of pSB1C3 was cut with EcoRI and PstI. The digestion mix ran on a agarose gel, after which gel purification of the linearized backbone occurred.
 
70ng of vector was used to ligate ssUSP45DspB.
 
The yield of transformants was very low after transforming the ligation mixture and growing the bacteria. Only one colony contained the ssUSP45DspB according to gel.