IGEM:UNAM Genomics Mexico/2009/Notebook/iGEM 2011/2011/04/12
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So, today, the team gathered and organized to do the four final constructions that are to be sent synthesized. Fibo and I took the first construction, the one with the fussion protein (HydA1-Fd). We assembled the parts and then checked for in silico mutations, for which I found none. We then looked for restriction enzymes sites that are forbiden in our constructions.
Standard Assembly: XbaI,PstI,SpeI,EcoRI,EcoRV,BamHI
Cut sites that are to be used to split parts: NdeI,AatII
Sites that are to be used to take off parts from plasmids: KpnI,SacI,AscI,PacI
It was first determined that the enzyme BglII was the one we were going to use to take off the poly-histidine tag. But as some of the members of the team got noticed, the codon adaptation index of the codons of this restriction site was way to low to be used and maintain the codon optimization throughout the sequence. We therefore opted to change the restriction enzyme to AatII, which did fullfill the requirements.