Part:BBa_K3944042
FabD
Bacterial fatty acid synthase (FAS) system enzyme, codon optimized for S.Cerevisiae. Part of the FAS2 system from E.coli, see our wiki for an overview of the system.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 34
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 618
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
FabD converts the primed malonate-CoA molecule into malonate-ACP, readying it for decarboxylation by FabH.
Overview map of FAS2 system (2).
Experimental design
By creating a single genetic fragment with FabD and FabZ and their promoters going in the forward and reverse direction, these two genes are easily inserted using the pCfB2909(XII-5 MarkerFree) plasmid from EasyClone kit (3).
Methodology
Double gene fragment ordered from IDT was cloned using Phusion polymerase according to protocol. Insert was purified following protocol for gel electrophoresis and ThermoFischer gel purification kit, followed by assembly into linearized plasmid backbone. The constructed plasmid was transformed into E.coli, grown overnight, inoculated overnight again, followed by harvesting following protocol for ThermoFischer plasmid miniprep kit. The purified plasmid was digested to yield the linearized insertion fragment and transformed into S.Cerevisiae strain CEN.PK 102-5B along with CAS9 and gRNA plasmids.
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