Difference between revisions of "Part:BBa K4447003"
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4447003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4447003 SequenceAndFeatures</partinfo>
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In our project, rifampicin monooxygenase <b>(EC 1.14.13.211)</b> is used as a detector for the presence of rifampicin by catalyzing the hydroxylation of rifampicin to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. As shown in <b>Figure 1</b>, this reaction requires NADPH as a reagent and, therefore, gives NADP+ as a reaction product. Consequently, it is possible to evaluate the presence of rifampicin through a coupled reaction employing a NADP+/NADPH colorimetric assay.
 
In our project, rifampicin monooxygenase <b>(EC 1.14.13.211)</b> is used as a detector for the presence of rifampicin by catalyzing the hydroxylation of rifampicin to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. As shown in <b>Figure 1</b>, this reaction requires NADPH as a reagent and, therefore, gives NADP+ as a reaction product. Consequently, it is possible to evaluate the presence of rifampicin through a coupled reaction employing a NADP+/NADPH colorimetric assay.
  
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[[Image:RifMo_reaction_TecMonterreyGDL.jpeg|600px|center|thumb|<b>Figure 1</b>. Chemical reaction for rifampicin monooxygenase (RifMo).]]
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[[Image:RifMo_reaction_TecMonterreyGDL.jpeg|650px|center|thumb|<b>Figure 1</b>. <i>Chemical reaction for rifampicin monooxygenase (RifMo).</i>]]
  
 
Rifampicin monooxygenase, as pictured below in <b>Figure 2</b> is a dimeric protein that has 474 amino acids in length and 51.4 kDa in weight (Hoshino et al., 2010). Koteva and collaborators (2018) reported a Michaelis constant of 12 µM for rifampicin, concluding it has a unique affinity for this substrate.
 
Rifampicin monooxygenase, as pictured below in <b>Figure 2</b> is a dimeric protein that has 474 amino acids in length and 51.4 kDa in weight (Hoshino et al., 2010). Koteva and collaborators (2018) reported a Michaelis constant of 12 µM for rifampicin, concluding it has a unique affinity for this substrate.
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[[Image:RifMo_rotating_TecMonterreyGDL.gif|230px|center|thumb|<b>Figure 2</b>. <i>Three-dimensional structure of RifMo.</i>]]
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=Characterization: TecMonterreyGDL 2024=
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== Cloning RifMo into pET28b vector ==
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As part of the development of our FRET-based biosensor for rifampicin [https://parts.igem.org/Part:BBa_K5439003 (BBa_K5439003)], we decided to clone the basic coding sequence into pET28b to contribute to the characterization and use of the basic part. After amplification by PCR and digestion with <i> NdeI </i> and <i> XhoI </i> <b> (Figure 3) </b>, the insert was ligated into the vector using T4 ligase (Invitrogen) at both a 3:1 and 5:1 molar ratio, using 50 ng of vector. The restriction digestion conditions are shown in <b> Table 1 </b>, while the ligation conditions are shown in <b> Table 2 </b>.
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{| class="wikitable" style="margin:auto; text-align:center; length: 80%"
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|+ Table 1. Restriction digest conditions.
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|-
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!Component !! Volume
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|-
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|-
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| style="text-align:center;" style="width: 80%;" | Restriction Enzyme 10X Buffer || 5 µL
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|--
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| style="text-align:center;" style="width: 80%;" | DNA (1 μg/μL) || 1 µL
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|-
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| style="text-align:center;" style="width: 80%;" | <i>NdeI</i> restriction enzyme|| 1 µL
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|-
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| style="text-align:center;" style="width: 80%;" | <i>XhoI</i> restriction enzyme || 1 µL
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|-
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| style="text-align:center;" style="width: 80%;" | BSA (10 μg/μL) || 0.2 µL
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|-
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| style="text-align:center;" style="width: 80%;" | Nuclease-free water || To 20 µL
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|-
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| style="text-align:center;" style="width: 80%;" | Total Volume || 20 µL
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|}
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{| class="wikitable" style="margin:auto; text-align:center; length: 60%"
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|+ Table 2. Ligation conditions.
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|-
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!Component !! 3:1 ratio !! 5:1 ratio
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|-
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| style="text-align:center;" style="width: 60%;" | pET28b || 50 ng (6.7 μL)  |||  50 ng (6.7 μL)
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|-
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| style="text-align:center;" style="width: 60%;" | RifMo || 39.74 ng (6.7 μL)  ||| 66.23 ng (11 μL)
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|--
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| style="text-align:center;" style="width: 60%;" | T4 ligase buffer || 2 μL ||| 2 μL
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|-
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| style="text-align:center;" style="width: 60%;" | T4 ligase || 0.2 μL ||| 0.2 μL
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|-
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| style="text-align:center;" style="width: 60%;" | Nuclease-free water || 4.4 μL |||  0 μL
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|}
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        <img src="https://static.igem.wiki/teams/5439/registry-parts/digestion-rifmo.webp" width="300">
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        <figcaption><b>Figure 3</b> Agarose gel electrophoresis of digested RifMo and digested pET28b samples.
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After ligation, the assembled product was transformed into <i> E. coli </i> BL21 by adding 5 μL of the ligation reaction to 50 μL of competent cells and transforming by heat shock. After incubation, there were some colonies visible in the case of the 3:1 ratio, as shown in <b> Figure 4 </b>
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        <img src="https://static.igem.wiki/teams/5439/registry-parts/transformadarifmo.webp" width="300">
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        <figcaption><b>Figure 4</b> Transformed colonies with pET28b + RifMo
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Finally, to confirm the presence of the insert in the transformed colonies, a colony PCR was performed with two colonies, where a band was observed near the 1500 bp mark, which matches the length of the gene (1422 bp) <b> (Figure 5) </b>
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        <img src="https://static.igem.wiki/teams/5439/registry-parts/cpcrrifmo.webp" width="300">
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        <figcaption><b>Figure 5</b>. Amplification of RifMo from 2 colonies corresponding to the 3:1 ligation ratio in BL21. The observed band, near the 1500 bp mark, matches the length of the gene.
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=References=
 
=References=

Latest revision as of 07:48, 2 October 2024


RifMo coding sequence

Rifampicin monooxygenase coding sequence from Nocardia farcinica. This enzyme catalyzes the oxidation of rifampicin, thereby inactivating its antibiotic activity. It constitutes a secondary rifampicin resistance factor.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1225
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1456
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Rifampicin is a potent antibiotic against tuberculosis and other mycobacterial infections, but an extensive usage of it and its derivatives has contributed to bacterial resistance, which neutralizes antibiotic activity. The presence of rifampicin in water bodies represents a persintent thread, because of the hazardous potential it has to aquatic organisms and human health (Cai et al., 2019).

In our project, rifampicin monooxygenase (EC 1.14.13.211) is used as a detector for the presence of rifampicin by catalyzing the hydroxylation of rifampicin to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. As shown in Figure 1, this reaction requires NADPH as a reagent and, therefore, gives NADP+ as a reaction product. Consequently, it is possible to evaluate the presence of rifampicin through a coupled reaction employing a NADP+/NADPH colorimetric assay.

Figure 1. Chemical reaction for rifampicin monooxygenase (RifMo).

Rifampicin monooxygenase, as pictured below in Figure 2 is a dimeric protein that has 474 amino acids in length and 51.4 kDa in weight (Hoshino et al., 2010). Koteva and collaborators (2018) reported a Michaelis constant of 12 µM for rifampicin, concluding it has a unique affinity for this substrate.

Figure 2. Three-dimensional structure of RifMo.


Characterization: TecMonterreyGDL 2024

Cloning RifMo into pET28b vector

As part of the development of our FRET-based biosensor for rifampicin (BBa_K5439003), we decided to clone the basic coding sequence into pET28b to contribute to the characterization and use of the basic part. After amplification by PCR and digestion with NdeI and XhoI (Figure 3) , the insert was ligated into the vector using T4 ligase (Invitrogen) at both a 3:1 and 5:1 molar ratio, using 50 ng of vector. The restriction digestion conditions are shown in Table 1 , while the ligation conditions are shown in Table 2 .

Table 1. Restriction digest conditions.
Component Volume
Restriction Enzyme 10X Buffer 5 µL
DNA (1 μg/μL) 1 µL
NdeI restriction enzyme 1 µL
XhoI restriction enzyme 1 µL
BSA (10 μg/μL) 0.2 µL
Nuclease-free water To 20 µL
Total Volume 20 µL


Table 2. Ligation conditions.
Component 3:1 ratio 5:1 ratio
pET28b 50 ng (6.7 μL) 50 ng (6.7 μL)
RifMo 39.74 ng (6.7 μL) 66.23 ng (11 μL)
T4 ligase buffer 2 μL 2 μL
T4 ligase 0.2 μL 0.2 μL
Nuclease-free water 4.4 μL 0 μL


Figure 3 Agarose gel electrophoresis of digested RifMo and digested pET28b samples. .

Figure 4 Transformed colonies with pET28b + RifMo .

Finally, to confirm the presence of the insert in the transformed colonies, a colony PCR was performed with two colonies, where a band was observed near the 1500 bp mark, which matches the length of the gene (1422 bp) (Figure 5)

Figure 5. Amplification of RifMo from 2 colonies corresponding to the 3:1 ligation ratio in BL21. The observed band, near the 1500 bp mark, matches the length of the gene. .


References

[1]. Cai, W., Weng, X., & Chen, Z. (2019). Highly efficient removal of antibiotic rifampicin from aqueous solution using green synthesis of recyclable nano-Fe3O4. Environmental pollution (Barking, Essex : 1987), 247, 839–846. https://doi.org/10.1016/j.envpol.2019.01.108

[2]. Hoshino, Y., Fujii, S., Shinonaga, H., Arai, K., Saito, F., Fukai, T., Satoh, H., Miyazaki, Y., & Ishikawa, J. (2010). Monooxygenation of rifampicin catalyzed by the rox gene product of Nocardia farcinica: structure elucidation, gene identification and role in drug resistance. The Journal of antibiotics, 63(1), 23–28. https://doi.org/10.1038/ja.2009.116

[3]. Koteva, K., Cox, G., Kelso, J. K., Surette, M. D., Zubyk, H. L., Ejim, L., Stogios, P., Savchenko, A., Sørensen, D., & Wright, G. D. (2018). Rox, a Rifamycin Resistance Enzyme with an Unprecedented Mechanism of Action. Cell Chemical Biology, 25(4), 403-412.e5. https://doi.org/10.1016/j.chembiol.2018.01.009

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