Composite

Part:BBa_K3711054

Designed by: Jiacheng Shi   Group: iGEM21_HUST-China   (2021-10-03)


Panb1-α factor-FMO-AOX1 Terminator


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 124
    Illegal XhoI site found at 649
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 706
    Illegal AgeI site found at 1925
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1928


Description

This is a composite part for extracellular expression of flavin-containing monooxygenase. FMO is expressed and participates in the production from indole to indigo.Panb1 is a constitutive promoter in yeast, which is expressed under anaerobic conditions, while under aerobic conditions, Panb1, as a repression target of ROX1, is inhibited. When Panb1 initiates the expression, the signal peptide, α-factor, is used to express FMO outside of the cell.

Usage and Biology

FMO is a kind of monooxygenase, which could produce C4a peroxyflavin intermediate, which is highly stable due to spectrum observation and could remain unchanged under 4℃ for minutes, even hours. In the first cycle, NADPH reduces FAD into FADH2. This reduced flavin could react with oxygen rapidly and become peroxyflavin, which is the main form of FMO and further react wih an appropriate nucleophilic substrate. This reaction will form one molecular of water and transfer one oxygen atom to the substrate. The release of NADP+ may be the rate-limiting step of catalytic cycle while the Vmax of substrate binding has little impact on both reactions.

Molecular cloning

Fig1. Plasmid construction and colony PCR results of Panb1-α factor-FMO-AOX1 Terminator and Pynr071c-α factor-ROX1-AOX1 Terminator transformed E.coli
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Categories
Parameters
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