Coding
fiatluxE

Part:BBa_K4239005

Designed by: Guillaume FULCONIS   Group: iGEM22_INSA_Lyon1   (2022-10-06)


Enhanced luciferase substrate forming unit fiatluxE


Description

fiatluxE is to be used with fiatluxC (BBa_K4239001) and fiatluxD (BBa_K4239002). It codes for a subpart fatty acid reductase. With the subparts encoded by fiatluxC and fiatluxD, they form a complex that recycles fatty acids to fatty aldehydes. Fatty aldehydes will be used as a substrate for the luciferase protein.

The system fiatluxC/fiatluxD/fiatluxE is made to be used with fiatluxA (BBa_K4239003) and fiatluxB (BBa_K4239004), gathered in the fiatluxCDABE (BBa_K4239008) operon.

fiatlux genes come from ilux genes (C, D, A, B, E). They were modified to remove every iGEM restriction site (EcoRI, XbaI, SpeI and PstI) included in genes. They were also adapted to include the biobrick format.

The ilux operon was born from a mutated natural luminescence operon present in the bacteria P.luminescens: the lux operon. These mutations were error-prone PCR induced according to Gregor et al.’s study in 2018 (Gregor et al. 2018). The aim was to create a system of genes that produced more light than the lux system.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 588
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Construction

The ilux operon was available in a pGEX plasmid. fiatluxA, fiatluxB and fiatluxE were directly constructed together in fiatluxABE. iGEM restriction sites were successfully removed in the iluxABE genes by following these steps: DNA extraction, PCR directed mutagenesis, agarose gel analysis with green gel, and gel purification. A classical PCR was performed to reconstitute iluxABE fragments which had been cut by the restriction enzymes. The part is now called fiatluxABE. This part was then cloned and transformed in a pSB1C3 (already in iGEM biobrick format) and pBAD18 (high-copy vector with an arabinose inducible promoter) plasmid in E.coli DH5α. More details about the construction are on the following page fiatluxABE (BBa_K4239007).

References

Gregor C, Gwosch KC, Sahl SJ, Hell SW. Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging. Proc Natl Acad Sci U S A. 2018 Jan 30;115(5):962-967. doi: 10.1073/pnas.1715946115. Epub 2018 Jan 16. PMID: 29339494; PMCID: PMC5798359.

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