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).

Cloning TjPCs insert into pET28b(+) vector

In order heterologously overexpress PCs in Escherichia coli, a ligation was carried out with TjPCs and a vector pET28b(+). This was achieved with T4 DNA ligase (Invitrogen), with 5:1 molar ratio following the protocol as observed in Table 1.

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).

Sequence and Features

References

[1]. 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

[2]. Jan-Roblero, J., & Cruz-Maya, J. A. (2023). Ibuprofen: toxicology and biodegradation of an emerging contaminant. Molecules, 28(5), 2097