Team:Groningen/Template/MODULE/Notebook/toolbox/week11

The sequences of NisA, NisC, NisRK and sfGFP(Bs) were analyzed. The sequences of NisA and sfGFP(Bs) were completely correct. The sequence of NisC showed that a mutation occured at residue 185, where an AAT was changed to an AAA, changing an asparagine to a lysine. The changed amino acid was on the outside of the protein, so may not have an effect on the function of NisC. The sequence of NisRK showed two mutations. At residue 199 of NisR a GAA was changed to a GAG, not changing the amino acid at this position. At residue 300 of NisK an ACA was changed to an ATA, changing a threonine to an isoleucine. The PNisI with RBS showed a large mutation. At position 508, 18 basepairs were missing. Therefore it was decided to not send PNisI to iGEM HQ anymore.

A PCR was done to generate fragments for assembling genes that had illegal restriction sites. Every fragment was succesfully amplified. The fragments were purified using the GeneJET PCR clean up kit. Not all fragments have a sufficient concentration or purity, so only the fragments for the combined BioBrick of NisF, NisE and NisG was made using Gibson assembly. The transformants that contained pSB1C3 with NisFEG were tested using colony PCR. No positive transformants were found.