Part:BBa_K4239008

fiatlux genes with their promoter to emit luminescence

Sequence and Features

BBa_K4239008 SequenceAndFeatures

Description

The fiatlux operon is composed of 6 genes: fiatluxA (BBa_K239003), fiatluxB (BBa_K239004), fiatluxC (BBa_K239001), fiatluxD (BBa_K239002) and fiatluxE (BBa_K239005), that form an autonomous system producing bioluminescence thanks to the encoded luciferase enzymes.

The genes fiatluxA and fiatluxB code each of them for a subunit of the luciferase protein. Both subunits need to be used together. Luciferase has as substrats FMNH2, O2 and Fatty aldehydes, and produces H20, Fatty Acids and FMN and emits luminescence.

The genes fiatluxC, fiatluxD and fiatluxE code each of them for a subpart of a fatty acid reductase. They need to be used together to form a complex that recycles fatty acids to fatty aldehydes. Fatty aldehydes will be used as a substrate for the luciferase protein.

Both systemes (fiatluxC/fiatluxD/fiatluxE and fiatluxA/fiatluxB) are made to be used together, and are gathered in the fiatluxCDABE operon.

Fiatlux genes come from ilux genes (C, D, A, B, E). They were modified to remove every Igem restriction site (EcoR1, Xba1, Spe1 and Pst1) 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.

Construction

Both parts fiatluxCD (BBa_K239006) and fiatluxABE (BBa_K239007) were created separated, as described on their own page. The next step was then to assemble both parts, to check that the operon was still functional after being mutated and reassembled, and to put it in an appropriate backbone to be able to transfer our fiatlux operon to other strains, even non-transformable ones, making our tool usable for more potential bacteria. Besides, we want to assemble J23117-fiatluxCD and fiatluxABE together in a conjugative plasmid, possessing an origin of transfer. An assembly of the J23117-fiatluxCD and fiatluxABE was thus done by HIFI assembly in pSEVA521 and pSEVA531, two conjugative vectors with RK2 and pBBR origin of replication respectively (low replication strength). These two plasmids were transformed in E. coli DH5α, and cloning was verified by a restriction profile. The ilux assembly of fiatluxABE and fiatluxCD was successfully done in both pSEVA521 and pSEVA531 vectors.

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.