Part:BBa_K4239008

fiatlux genes with their promoter to emit luminescence

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 systems (fiatluxC/fiatluxD/fiatluxE and fiatluxA/fiatluxB) are 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 (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
  INCOMPATIBLE WITH RFC[12]

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

  Illegal BglII site found at 5342
  Illegal BamHI site found at 50
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2993
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1366
  Illegal SapI site found at 3512
  

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.

Characterization

Our fiatlux tool was designed in order to achieve a perfect balance between luminescence output and regular bacterial growth. It is critical that the presence of our plasmid does not disrupt the regular functioning of the transformed bacteria; otherwise, an imaging study using our technology would be inaccurate. A bacterial growth defect due to our fiatluxCDABE system could impact bacterial processes such as virulence. In order to determine the appropriate culture and storage conditions, it is also necessary to characterize how the transformed strains react to temperature changes after cold storage and various antibiotic concentrations.

As an initial stage, optimization and characterization were carried out in E. coli DH5. We have created 2 plasmids that differ only in their origin of replication. pSEVA521-fiatluxCDABE carries out the RK2 replication origin whereas pSEVA531-fiatluxCDABE has the pBBR1 replication origin. The number of copies of these plasmids were not evaluated in our studies but based only on previous studies performed in E. coli (Jahn et al. 2016) that evaluated a copy number between 2 and 7 for RK2-based plasmid and between 5 to 10 from pBBR1 replication origin. The effect on E. coli of the fiatluxCDABE expression was evaluated by comparing the phenotypes of E. coli strains expressing fiatluxCDABE with those carrying the empty vector pSEVA521 or pSEVA531.

Effect of Tetracycline on the Growth and Luminescence of E. coli fiatluxCDABE Strains on Solid Media.

First, we analyzed the behavior of the fiatluxCDABE strains on solid LB media. We streaked E. coli strains on LB agar containing various concentrations of tetracycline. Because we observed that DH5α with pSEVA531-fiatluxCDABE streaked on a LB agar plate with tetracycline at 10 µg/ml had difficulties to grow, we decided to determine the effect of tetracycline on the strains containing pSEVA521-fiatluxCDABE, pSEVA531-fiatluxCDABE or the empty vectors. We also decided to evaluate their growth depending on storage conditions. Strains were directly streaked from the -80°C glycerol stock, from colonies stored at 4°C or at room temperature. The plates were then incubated at 37°C overnight. Results are presented in Figure 1.

Transformed E. coli DH5α were able to grow after being stored at -80°C or +4°C, except in the presence of a 10 µg/mL tetracycline medium. When stored at room temperature, transformed E. coli DH5α were able to grow at any tetracycline concentration. We also observed that DH5α transformed with pSEVA521-fiatlux grew better in a 2 and 5 µg/mL tetracycline LB agar medium. In contrast, DH5α strains containing the empty vectors pSEVA521 or pSEVA531 grew very well on LB agar with 10 µg/ml tetracycline, even when the inoculum is from bacteria stored at 4°C or -80°C. Taken together, these observations indicate that expression of fiatlux from pSEVA531 certainly delays the ability of the E. coli DH5α to resist an usual concentration of tetracycline since the use of lower concentrations of tetracycline improves growth. It is likely that the production of light by luciferase has an energy cost that would prevent metabolically inactive bacteria (as stored at 4°C or -80°C) from adapting to the presence of tetracycline.

Figure 1.A - A: E.coli transformed with pSEVA531-J23117-fiatluxCDABE, B: E.coli transformed with pSEVA531-J23117-fiatluxCDABE, C: E.coli transformed with an empty pSEVA531, D: E. coli WT.
Antibiotics and temperature characterizations of E.coli strains transformed with fiatlux (A and B). For each tetracycline concentration (2, 5 or 10 µg/mL), a different storage temperature was tested (-80°C, 4°C or RT), and the effect on luminescence was observed.

Figure 1.B - Same Petri dishes as Figure 1.A but with luminescence. Pictures taken with NightSHADE LB 985 In Vivo Plant Imaging System

From now on, a tetracycline concentration between 2 and 5 µg/ml should be used for E.coli DH5α transformed strains culture.

Effect of Tetracycline on the Growth and Luminescence of E.coli fiatluxCDABE Strains in Liquid Media.

We also compared the growth over time (O.D.) of E. coli DH5a containing pSEVA521, pSEVA531, pSEVA521-fiatluxCDABE and pSEVA531-fiatluxCDABE, with a TECAN microplate reader. Luminescence was also measured over time. Bacteria were cultivated in LB at 37°C with shaking microplates. To note that we concentrated our bio modelisation analysis on Dickeya solani behavior and not on E.coli’s. Thus, all of our E.coli graphics and results are not statistically confirmed but based on observation, owing to time constraints.

Figure 2 - O.D. of E.coli transformed strains with pSEVA521 vectors in function of time. Blue data correspond to pSEVA521-fiatlux transformed strains, red data correspond to empty pSEVA521 transformed strains. All data have different shapes: crosses correspond to tet0µg/mL, squares to 5µg/mL and triangles to 10µg/mL. In this graphic, only one replicate is shown among the 16 performed replicates, for each condition. The represented replicate is representative of the global results.

Figure 3 - O.D. of E.coli transformed strains with pSEVA531 vectors in function of time. Blue data correspond to pSEVA531-fiatlux transformed strains, red data correspond to empty pSEVA531 transformed strains. All data have different shapes: crosses correspond to tet0µg/mL, squares to 5µg/mL and triangles to 10µg/mL. In this graphic, only one replicate is shown among the 16 performed replicates, for each condition. The represented replicate is representative of the global results.

In this graphic, only one replicate is shown among the 16 performed replicates, for each condition. The represented replicate is representative of the global results.

Concerning pSEVA521 transformed strains, fiatlux seems to slightly affect the maximum O.D., but a statistical analysis would be necessary to confirm its significance.
Concerning pSEVA531 transformed strains, pSEVA531-fiatlux transformed bacteria and empty pSEVA531 transformed bacteria in Tet 0µg/mL seems to have similar maximum O.D., whereas a growth lag seems to occur in Tet 10µg/mL transformed bacteria. Indeed, the beginning of growth is visible starting from 7 000 secs for empty pSEVA531 transformed bacteria, and from 75 000 secs for pSEVA531-fiatlux transformed bacteria. Therefore, it seems that pSEVA521 is a better vector to use in E.coli.

Then, the luminescence over growth ratio is analyzed to understand how fiatlux transformed E.coli bacteria behave, in function of the used vector (pSEVA531 or 521) and in function of the antibiotic concentration (tet 10, 5 or 0 µg/mL).

Figure 4 - Luminescence over O.D. ratio of E.coli transformed strains with pSEVA521 vectors as a function of time. Blue data correspond to pSEVA521-fiatlux transformed strains, red data correspond to empty pSEVA521 transformed strains. All data have different shapes: crosses correspond to tet0µg/mL, squares to 5µg/mL and triangles to 10µg/mL. In this graphic, only one replicate is shown among the 16 performed replicates, for each condition. The represented replicate is representative of the global results.

Figure 5 - Luminescence over O.D. ratio of E.coli transformed strains with pSEVA531 vectors in function of time. Blue data correspond to pSEVA531-fiatlux transformed strains, red data correspond to empty pSEVA531 transformed strains. All data have different shapes: crosses correspond to tet0µg/mL, squares to 5µg/mL and triangles to 10µg/mL. In this graphic, only one replicate is shown among the 16 performed replicates, for each condition. The represented replicate is representative of the global results.

These graphics show that, whatever the tetracycline concentration is, E.coli DH5alpha transformed with fiatlux emits luminescence, and E.coli bacteria that do not have our fiatlux operon do not produce any light. It is interesting to note that, in a tet 0µg/mL growth medium, there is no selective pressure on the transformed bacteria anymore, but these bacteria were still shown to produce luminescence. Besides, for all antibiotics concentration, pSEVA521-fiatlux transformed bacteria’s behavior seems similar, whereas pSEVA531-fiatlux transformed bacteria’s behavior is irregular, hence not reproducible.

To note that the luminescence is comparable between pSEVA521-fiatlux transformed bacteria and pSEVA531-fiatlux transformed bacteria. Besides, by comparing both O.D and luminescence/O.D. ratio graphics, the maximum luminescence emission is observed at the end of the exponential growth phase, which confirms that luminescence is potentially dependent on the internal bacterial metabolism.

In the end, both liquid and solid media showed that the plasmid pSEVA521 seemed more suitable than pSEVA531 in E.coli DH5α strains. We believe that the reason could be the following: a higher replication rate could tire the bacteria and thus slow down its growth, which could be due to the potential toxic effect the high production of luminescence could have on the bacteria.

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.

Jahn, M., Vorpahl, C., Hübschmann, T. et al. Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR. Microb Cell Fact 15, 211 (2016). https://doi.org/10.1186/s12934-016-0610-8