Coding

Part:BBa_K3944040

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


FabB

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
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 885
    Illegal SpeI site found at 898
    Illegal PstI site found at 1103
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 885
    Illegal NheI site found at 66
    Illegal SpeI site found at 898
    Illegal PstI site found at 1103
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 885
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 885
    Illegal SpeI site found at 898
    Illegal PstI site found at 1103
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 885
    Illegal SpeI site found at 898
    Illegal PstI site found at 1103
    Illegal NgoMIV site found at 559
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Responsible for the elongation step in fatty acid synthesis, adding another two carbons onto the growing chain and continuing the cycle from initiation or continuation. See FabG for the next step.

FAS2 Chalms Gu 2021.png

Overview map of FAS2 system (2).

Experimental design

By creating a single genetic fragment with FabB and AcpP 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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