Difference between revisions of "Part:BBa K5439005"
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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). | 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 | + | =Cloning ipfF insert into pET28b(+) vector= |
| − | In order heterologously overexpress PCs in <i>Escherichia coli</i>, a ligation was carried out with | + | In order heterologously overexpress PCs in <i>Escherichia coli</i>, a ligation was carried out with ipfF and a vector pET28b(+). This was achieved with T4 DNA ligase (Invitrogen), with 5:1 molar ratio following the protocol as observed in <b>Table 1</b>. |
{| class="wikitable" style="margin:auto; text-align:center; length: 80%" | {| class="wikitable" style="margin:auto; text-align:center; length: 80%" | ||
| − | |+ Table 1. Ligation of | + | |+ Table 1. Ligation of ipfF insert and pET28b(+) vector (5:1 molar ratio). |
|- | |- | ||
!Reagent !! Volume (µL) 5:1 ratio | !Reagent !! Volume (µL) 5:1 ratio | ||
| Line 27: | Line 27: | ||
| style="text-align:center;" style="width: 80%;" | pet28b(+) || 6.7 µL | | style="text-align:center;" style="width: 80%;" | pet28b(+) || 6.7 µL | ||
|- | |- | ||
| − | | style="text-align:center;" style="width: 80%;" | | + | | style="text-align:center;" style="width: 80%;" | ipfF || 2.8 µL |
|-- | |-- | ||
| style="text-align:center;" style="width: 80%;" | T4 DNA Ligase Buffer || 2 µL | | style="text-align:center;" style="width: 80%;" | T4 DNA Ligase Buffer || 2 µL | ||
| Line 33: | Line 33: | ||
| style="text-align:center;" style="width: 80%;" | T4 DNA ligase|| 0.2 µL | | style="text-align:center;" style="width: 80%;" | T4 DNA ligase|| 0.2 µL | ||
|- | |- | ||
| − | | style="text-align:center;" style="width: 80%;" | Nuclease-free water|| | + | | style="text-align:center;" style="width: 80%;" | Nuclease-free water|| 8.3 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 <b>Figure 1</b>, incubated overnight at 37 ºC in LB agar and kanamycin (50 μg/mL). | 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 <b>Figure 1</b>, incubated overnight at 37 ºC in LB agar and kanamycin (50 μg/mL). | ||
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[1]. Murdoch, R. W., & Hay, A. G. (2013). Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2. <i>Microbiology (Reading, England)</i>, 159(Pt 3), 621–632. https://doi.org/10.1099/mic.0.062273-0 | [1]. Murdoch, R. W., & Hay, A. G. (2013). Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2. <i>Microbiology (Reading, England)</i>, 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. <i>Molecules</i>, 28(5), 2097 | + | [2]. Jan-Roblero, J., & Cruz-Maya, J. A. (2023). Ibuprofen: toxicology and biodegradation of an emerging contaminant. <i>Molecules</i>, 28(5), 2097. https://doi.org/10.3390/molecules28052097 |
| + | |||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K5439005 parameters</partinfo> | <partinfo>BBa_K5439005 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
Revision as of 01:13, 2 October 2024
IpfF coding sequence
Long-chain fatty acid CoA ligase from Sphingomonas spp. This enzyme catalyzes the conversion of ibuprofen into isobutylcatechol.
Sequence and Features IpfF coding sequence
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).
Cloning ipfF insert into pET28b(+) vector
In order heterologously overexpress PCs in Escherichia coli, a ligation was carried out with ipfF 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.
| Reagent | Volume (µL) 5:1 ratio |
|---|---|
| pet28b(+) | 6.7 µL |
| ipfF | 2.8 µL |
| T4 DNA Ligase Buffer | 2 µL |
| T4 DNA ligase | 0.2 µL |
| Nuclease-free water | 8.3 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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1592
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 463
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1460
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. https://doi.org/10.3390/molecules28052097