Coding

Part:BBa_K3944041

Designed by: Olof Dahlman   Group: iGEM21_Chalmers-Gothenburg   (2021-09-30)


FabH

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 213
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

FabH catalyses the decarboxylation of the terminal carboxyl group to prepare the initiated chain for elongation by FabB. See FabB for the next step.

FAS2 Chalms Gu 2021.png

Overview map of FAS2 system (2).

Experimental design

By creating a single genetic fragment with FabH and AcpS and their promoters going in the forward and reverse direction, these two genes are easily integrated into the yeast genome using the pCfB3040(XII-4 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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