Part:BBa_K3944038
AcpP
Bacterial fatty acid synthesis (FAS) system enzyme AcpP, codon optimized for S.Cerevisiae (1,2). 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]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This enzyme codes for apo-apc, a precursor protein in fatty acid synthesis. The reaction catalysed prepares the starting block for subsequent conversion by AcpS. See AcpS for the next step.
Overview map of bacterial FAS2 system (2).
Experimental design
By creating a single genetic fragment with AcpP and FabB and their promoters going in the forward and reverse direction, these two genes are easily integrated into the yeast genome using the pCfB3038(XII-1 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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