Part:BBa_K4239006

Enhanced luciferase substrate forming subunits fiatluxCD and the promoter J23117

Description

fiatluxC (BBa_K4239001) and fiatluxD (BBa_K4239002) are to be used with fiatluxE (BBa_K4329005). They code for a fatty acid reductase. With the subparts encoded by fiatluxE, 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 to be used with fiatluxA (BBa_K4239003) and fiatluxB (BBa_K4239004), gathered in the fiatluxCDABE operon (BBa_K4239008). fiatluxA and fiatluxB code for the luciferase protein

.

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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 50
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1366
  

Construction

fiatluxCD was directly synthesized with the promoter J23117 (BBa_J23117). It is a constitutive and weak promoter, so no inducer needs to be added. fiatluxCD and its promoter were ordered in the plasmid pACYDuet-1, a p15A low-copy plasmid, and transformed it into E.coli DH5α. fiatluxCD and the promoter J23117 are with the iGEM format (between EcoRI, XbaI, SpeI and PstI).

Figure 1: Construction of fiatluxCD

pACYDuet-1-J23117-fiatluxCD was requested because its origin of replication (p15A) and its resistance to chloramphenicol makes it compatible in the same bacteria with the plasmid pBAD18-fiatluxABE (BBa_K4239007) (ori colE1) which carries the resistance to kanamycin. The goal is to insert both plasmids in the same bacteria, to gather the entire fiatluxCDABE operon.

Characterization

To check that the synthesized DNA contains the genes fiatluxCD and the promotor, we carry out a miniprep of the plasmid pACYDuet-1-J23117-fiatluxCD. This last one is digested by two enzymes, EcoR1 and Pst1 (iGEM restriction sites), and a migration on agarose gel was performed. The expected fragments are:

• Non Digested plasmid: 6488 bp
• Double Digested plasmid: 2509 bp + 3979 bp

The strip at 2509 bp indicates that fiatluxCD and the promoter J23117 are indeed in the plasmid.

Figure 1: Construction of fiatluxCD

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.