Coding

Part:BBa_K5439005

Designed by: Diego Cota Barocio, Adhara Renata Cruz Tenorio   Group: iGEM24_TecMonterreyGDL   (2024-09-30)
Revision as of 02:02, 2 October 2024 by Adhara-renata04 (Talk | contribs)


IpfF coding sequence

Long-chain fatty acid CoA ligase from Sphingomonas spp. This enzyme catalyzes the conversion of ibuprofen into isobutylcatechol.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1592
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 463
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1460

Usage and Biology

Ibuprofen is an anti-inflammatory treatment drug widely used in the world that can be bought without any necessary prescription. This makes ibuprofen a drug that everyone can consume easily, bringing problems because its disposal makes it an emerging contaminant in water bodies 10. An example of it is Sphingomonas Ibu-2; an organism that has been grown in an environment rich in ibuprofen. The described organism has the ability to metabolize ibuprofen to isobutylcatechol due to the adaptation, which one particular gene is in charge of this degradation which is IpfF (Murdoch et al., 2013).

The gene ipfF participates in the lower ibuprofen degradation pathway, this gene encodes a CoA ligase enzyme which attaches CoA to ibuprofen (Jan-Roblero et al., 2023).

Characterization

To obtain a prediction of the ipfF structure using the coding sequence for the protein, it was used ColabFold (Jumper et al., 2021) and also used the best Predicted Aligned Error (PAE) (Figure1).

Figure 1. Predicted structure with the best PAE obtained from ColabFold showing the modeled ipfF sequence.

Cloning ipfF insert into pET28b(+) vector

In order heterologously overexpress ipfF in Escherichia coli, a ligation was carried out with ipfF and a vector pET28b(+). This was achieved with T4 DNA ligase (Invitrogen), with 3:1 molar ratio following the protocol as observed in Table 1.


Table 1. Ligation of ipfF insert and pET28b(+) vector (3:1 molar ratio).
Reagent Volume (µL) 3:1 ratio
pet28b(+) 6.7 µL
ipfF 1.7 µL
T4 DNA Ligase Buffer 2 µL
T4 DNA ligase 0.2 µL
Nuclease-free water 9.4 µL

After 1 hour incubation at 22 ºC, the resulting ligation was transformed through heat shock in E.coli BL21 chemically competent cells. The successful results from the transformation can be noted in Figure 1, incubated overnight at 37 ºC in LB agar and kanamycin (50 μg/mL).

Figure 2. Transformation of pET28b(+)_ipfF plasmid into E. coli BL21 cells.


References

[1]. Jan-Roblero, J., & Cruz-Maya, J. A. (2023). Ibuprofen: toxicology and biodegradation of an emerging contaminant. Molecules, 28(5), 2097. https://doi.org/10.3390/molecules28052097

[2]. Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., Bridgland, A., Meyer, C., Kohl, S. A. A., Ballard, A. J., Cowie, A., Romera-Paredes, B., Nikolov, S., Jain, R., Adler, J., … Hassabis, D. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 596(7873), 583–589. https://doi.org/10.1038/s41586-021-03819-2

[3]. Murdoch, R. W., & Hay, A. G. (2013). Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2. Microbiology (Reading, England), 159(Pt 3), 621–632. https://doi.org/10.1099/mic.0.062273-0

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