Part:BBa_K4239007

Enhanced units fiatluxABE

Description

fiatluxA (BBa_K4239003) and fiatluxB (BBa_K4239004) are to be used together. They code for the luciferase protein.

Luciferase has as substrates FMNH₂, O₂ and Fatty aldehydes, and produces H₂0, Fatty Acids and FMN and emits luminescence.

The system fiatluxA/fiatluxB is to be used with fiatluxC, fiatluxD and fiatluxE (BBa_K4239006), 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 (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.

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 2853
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 504
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1023
  

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 in the following plasmids, which were then transformed into E.coli DH5α:

• : in a pSB1C3 (already in iGEM biobrick format), without promotor
• : in a pBAD18, under the control of the arabinose inductible promotor pBAD.

pBAD18-fiatluxABE has a kanamycin resistance gene, which is used to select clones with the plasmid.

Figure 1: Construction of fiatluxABE

Characterization

1. Verify the plasmids' construction

To check that the new plasmids contains the genes fiatluxABE, we carry out a miniprep of the plasmid pBAD18-fiatluxABE. 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: 8833 bp
• Double Digested plasmid: 3439 bp + 5394 bp

The strip at 3439 bp indicates that fiatluxABE is in the plasmid.

Figure 2: Migration on agarose gel of the plasmid pBAD18-fiatluxABE

2. Effect of silent mutations on the luminescence

Since mutations have been introduced in the ilux operon, the effect on bioluminescence had to be tested before reassembling fiatluxABE and fiatluxCD in the same plasmid. In order to check the bioluminescence, the two plasmids pACYDuet-1-fiatluxCD and pBAD18-fiatluxABE were inserted into E.coli DH5α by electrotransformation. After transformation, the bioluminescence of the transformants was well observed with ChemiDoc XRS+ (Bio-Rad highly-sensitive camera) indicating that the genes fiatluxABE and fiatluxCD are expressed and functional (Figure 3).

Figure 3: Luminescent E.coli DH5α containing pACYDuet-1-fiatluxCD and pBAD18-fiatluxABE captured with a highly-sensitive camera, without needing to add arabinose to induce the promoter (basal expression). Left: captured luminescence. Right: simple photo of the Petri dish

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.