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    <title>LC-MS of NicX wild type</title>
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    <table border="1">
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            <th>Picture-protein name-<br/>nicotine concentration-reaction time</th>
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            <th>[S]/ng</th>
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            <th>Δ[S]/ng</th>
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            <th>Δ[S] in origin/ng</th>
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            <th>Δ[S] in μmol</th>
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            <th>V/μmol·<p>min<sup>-1</sup></p></th>
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            <td>NicX 400μM 0.5</td>
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            <td>0.075</td>
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            <td>0.018</td>
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            <td>180</td>
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            <td>1.16</td>
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            <td>0.039</td>
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        </tr>
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            <td>NicX 400μM 1</td>
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            <td>0.057</td>
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            <td>0.018</td>
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            <td>180</td>
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            <td>1.16</td>
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            <td>0.039</td>
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'''Table 1.''' LC-MS for NicX wild type
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| '''Figure 12.''' NicX 400μM 0.5
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| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/icms-nicx-400-1.png></html>
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| '''Figure 13.''' NicX 400μM 1
 
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===References===
 
===References===

Revision as of 15:26, 11 October 2023

NicX

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 762
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 762
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 762
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 762
    Illegal NgoMIV site found at 921
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

The enzyme NicX, derived from the bacterium Bacteroides xylanisolvens[1][2], exhibits a predicted core structure akin to the computational model of the established nicotine-degrading enzyme, NicA. Notably, NicX demonstrates proficiency in the degradation of nicotine. Furthermore, it has been observed that in the presence of NicX, B. xylanisolvens exhibits an enhanced capacity to degrade nicotine. Moreover, the transferability of NicX into Escherichia coli has been demonstrated, with the DNA fragment encoding the full-length NicX gene being successfully cloned into the pET28a vector through conventional molecular cloning techniques (Pro-cet-cell). This particular NicX component serves as a fundamental element in the construction of our composite part denoted as J1-NicX[3].

Design Consideration

The genetic construct was ligated into a pET28a plasmid vector and subsequently introduced into Escherichia coli strain BL21 (DE3). Enzymatic cleavage was performed at the NcoI and XhoI restriction sites, allowing for the precise integration of NicX. The original His tag on the plasmid was retained, which is useful for subsequent protein purification steps. To enhance the stability of NicX within the human body, the J1 fusion protein was strategically linked in front of them. Specific point mutations were introduced to the NicX gene sequence with the aim of enhancing its enzymatic activity. NicX was genetically connected with other components to enable its direct translation onto the surface of BL21. This innovation eliminated the need for protein purification steps, allowing for the direct utilization of E. coli as a host for enzymes in various applications[4][5][6].

Protein Expression

Figure 1. (a) SDS-PAGE of INPNC- NicX- histag(1989bp) & NicX(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 1:INPNC- NicX- histag(1989bp)Before induction 2, 3, 4, 5, 6:After induction; 2: 37℃ 0.3mM IPTG,3: 37℃ 0.5mM IPTG,4: 37℃ 0.7mM IPTG,5: 37℃ 1mM IPTG 6: NicX(1293bp) Before induction 7,8:After induction; 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG (b) 1: 37℃ INPNC- NicX- histag(1989bp)Before induction 2-6:After induction; 2: 37℃ 0.3mM IPTG,3: 37℃ 0.5mM IPTG,4: 37℃ 0.7mM IPTG,5: 37℃ 1mM IPTG；6: 37℃ NicX(1293bp) Before induction 7-8:After induction; 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG
Figure 2. (a) SDS-PAGE of INPNC- NicX-histag(1989bp) & NicX(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 3: NicX (1293bp) Before induction 1,2: After induction; 1: 37℃ 0.5mM IPTG, 2: 37℃ 0.3mM IPTG 8:INPNC- NicX- histag(1989bp) Before induction 4,5,6,7:After induction; 4: 37℃ 1mM IPTG, 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG,7: 37℃ 0.3mM IPTG(b)3: 37℃ NicX (1293bp) Before induction 1-2: After induction; 1: 37℃ 0.5mM IPTG, 2: 37℃ 0.3mM IPTG 8: 37℃ INPNC- NicX- histag(1989bp) Before induction 4-7:After induction; 4: 37℃ 1mM IPTG, 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG,7: 37℃ 0.3mM IPTG
Figure 3. (a) SDS-PAGE of J1-Δ50NicA2(1458bp)&J1-NicX(1446bp)&J1-NicX(1446bp) &Δ50NicA2(1305bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: Δ50NicA2 (1305bp)Supernatant 3: J1-Δ50NicA2 (1458bp) Before Induction 2: After induction; 2: 37℃ 0.5mM IPTG 5: NicX(1293bp) Before induction 4: After induction; 37℃ 0.5mM IPTG 7: J1-NicX(1446bp) Before induction 6: After induction; 6: 37℃ 0.5mM IPTG 9: Δ50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM IPTG (b) 1: Δ50NicA2 (1305bp)Supernatant 3: 37℃ J1-Δ50NicA2 (1458bp) Before Induction 2: After induction; 2: 37℃ 0.5mM IPTG 5: 37℃ NicX(1293bp) Before induction 4: After induction; 37℃ 0.5mM IPTG 7: 37℃ J1-NicX(1446bp) Before induction 6: After induction; 6: 37℃ 0.5mM IPTG 9: 37℃ Δ50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM IPTG
Figure 4. (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h

M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: NicX-W52G(1293bp)Supernatant 2: Δ50NicA2 (1305bp)Washing buffer 3:NicX-W52G(1293bp)Washing buffer 4: NicX(1293bp)Washing buffer 8: LppOmpA-linker-NicX-histag(1770bp) Before induction 5,6,7: After induction; 5: 37℃ 0.5mM IPTG,6: 37℃ 0.7mM IPTG,7: 37℃ 0.1mM IPTG 9: NicX(1293bp)Supernatant (b)1: NicX-W52G(1293bp)Supernatant 2: Δ50NicA2 (1305bp)Washing buffer 3:NicX-W52G(1293bp)Washing buffer 4: NicX(1293bp)Washing buffer 8: 37℃ LppOmpA-linker-NicX-histag(1770bp) Before induction 5-7: After induction; 5: 37℃ 0.5mM IPTG,6: 37℃ 0.7mM IPTG,7: 37℃ 0.1mM IPTG 9:NicX(1293bp)Supernatant

Figure 5. (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h

M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: J1-Δ50NicA2 (1458bp) 2: Δ50NicA2 (1305bp)Supernatant 3:NicX-W52G(1293bp)Supernatant 4: NicX-V16G (1293bp)Supernatant 5: J1-NicX (1446bp) 6: NicX(1293bp) 7,8,9: LppOmpA-linker-NicX-histag(1770bp) After induction; 7: 37℃ 0.1mM IPTG,8: 37℃ 0.3mM IPTG,9: 37℃ 0.5mM IPTG 10: J1-Δ50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)Supernatant (b) 1: J1-Δ50NicA2 (1458bp) 2: Δ50NicA2 (1305bp)Supernatant 3:NicX-W52G(1293bp)Supernatant 4: NicX-V16G (1293bp)Supernatant 5: J1-NicX (1446bp) 6: NicX(1293bp) 7-9: LppOmpA-linker-NicX-histag(1770bp) After induction; 7: 37℃ 0.1mM IPTG,8: 37℃ 0.3mM IPTG,9: 37℃ 0.5mM IPTG 10: J1-Δ50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)Supernatant

Figure 6. (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h

M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: NicX-V16G(1293bp)Washing buffer 2: LppOmpA-linker-NicX-histag(1770bp) Before induction 3,4,5,6,7,8,9,: After induction; 3: 16℃ 0.3mM IPTG,4: 16℃ 0.5mM IPTG,5: 16℃ 0.7mM IPTG, 6: 37℃ 0.1mM IPTG, 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG,9: 37℃ 0.7mM IPTG 10: NicX-W52G(1293bp)Washing buffer 11: J1-Δ50NicA2 (1458bp)Washing buffer 12: NicX(1293bp)Washing buffer 13: J1-NicX (1446bp)Washing buffer 14: J1-Δ50NicA2 (1458bp)Washing buffer

Figure 7. (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) & NicX-W52G(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 1: LppOmpA-linker-NicX-histag(1770bp)Before induction 2, 3, 4:After induction; 2: 37℃ 0.7mM IPTG, 3: 37℃ 0.5mM IPTG, 4: 37℃ 0.3mM IPTG, 7,10: NicX-W52G(1293bp) Before induction 5,6,8,9:After induction; 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG, 8: 16℃ 0.7mM IPTG, 9: 16℃ 0.5mM IPTG (b) 1: LppOmpA-linker-NicX-histag(1770bp)Before induction 2-4:After induction; 2: 37℃ 0.7mM IPTG, 3: 37℃ 0.5mM IPTG, 4: 37℃ 0.3mM IPTG, 7: 37℃ NicX-W52G(1293bp) Before induction 5-6:After induction; 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG 10: 16℃ NicX-W52G(1293bp) Before induction 8-9:After induction; 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG, 8: 16℃ 0.7mM IPTG, 9: 16℃ 0.5mM IPTG
Figure 8. (a) SDS-PAGE of NicX-L48Q (1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 6,12: Before induction 1,2,3,4,5,7,8,9,10,11:After induction; 1: 37℃ 1mM IPTG,2: 37℃ 0.7mM IPTG,3: 37℃ 0.5mM IPTG, 4: 37℃ 0.3mM IPTG, 5: 37℃ 0.1mM IPTG, 7: 16℃ 1mM IPTG, 8: 16℃ 0.7mM IPTG, 9: 16℃ 0.5mM IPTG, 10: 16℃ 0.3mM IPTG, 11: 16℃ 0.1mM IPTG (b)6: 37℃ Before induction 1-5:After induction; 1: 37℃ 1mM IPTG,2: 37℃ 0.7mM IPTG,3: 37℃ 0.5mM IPTG, 4: 37℃ 0.3mM IPTG, 5: 37℃ 0.1mM IPTG 12: 16℃ Before induction 7-11:After induction; 7: 16℃ 1mM IPTG, 8: 16℃ 0.7mM IPTG, 9: 16℃ 0.5mM IPTG, 10: 16℃ 0.3mM IPTG, 11: 16℃ 0.1mM IPTG
Figure 9. (a) SDS-PAGE of NicX-Y49L(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 1,7:Before induction 2,3,4,5,6,8,9,10,11,12: After induction; 2: 16℃ 0.1mM IPTG, 3: 16℃ 0.3mM IPTG, 4: 16℃ 0.5mM IPTG, 5: 16℃ 0.7mM IPTG, 6: 16℃ 1mM IPTG, 8: 37℃ 0.1mM IPTG, 9: 16℃ 0.3mM IPTG, 10: 16℃ 0.5mM IPTG, 11: 16℃ 0.7mM IPTG, 12: 37℃ 1mM IPTG (b) 1: 16℃Before induction 2-6:After induction; 2: 16℃ 0.1mM IPTG,3: 16℃ 0.3mM IPTG, 4: 16℃ 0.5mM IPTG, 5: 16℃ 0.7mM IPTG, 6: 16℃ 1mM IPTG, 7: 16℃Before induction 8-12:After induction;8: 37℃ 0.1mM IPTG, 9: 16℃ 0.3mM IPTG, 10: 16℃ 0.5mM IPTG, 11: 16℃ 0.7mM IPTG, 12: 37℃ 1mM IPTG

LC-MS Data Analysis and Discussion

Figure 10. LC-MS experiment conditions 1
Figure 11. LC-MS experiment conditions 2

LC-MS of NicX wild type

Picture-protein name-
nicotine concentration-reaction time		 [S]/ng Δ[S]/ng Δ[S] in origin/ng Δ[S] in μmol V/μmol·

min-1
NicX 400μM 0.5 0.075 0.018 180 1.16 0.039
NicX 400μM 1 0.057 0.018 180 1.16 0.039
Table 1. LC-MS for NicX wild type


Figure 12. NicX 400μM 0.5


Figure 13. NicX 400μM 1

References

  1. ↑ Chen, B., Sun, L., Zeng, G., Shen, Z., Wang, K., Yin, L., ... & Jiang, C. (2022). Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature, 610(7932), 562-568. https://doi.org/10.1038/s41586-022-05299-4
  2. ↑ Jiménez, J. I., Canales, Á., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J. L., & Díaz, E. (2008). Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440. Proceedings of the National Academy of Sciences, 105(32), 11329-11334.https://doi.org/10.1073/pnas.080227310
  3. ↑ Xue, S., Kallupi, M., Zhou, B., Smith, L. C., Miranda, P. O., George, O., & Janda, K. D. (2018). An enzymatic advance in nicotine cessation therapy. Chemical Communications, 54(14), 1686-1689.DOI https://doi.org/10.1039/C7CC09134F
  4. ↑ Wang, S. N., Liu, Z., Tang, H. Z., Meng, J., & Xu, P. (2007). Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16. Microbiology, 153(5), 1556-1565. https://doi.org/10.1099/mic.0.2006/005223-0
  5. ↑ Wang, W., Xu, P., & Tang, H. (2015). Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste. Scientific Reports, 5(1), 16411. https://doi.org/10.1038/srep16411
  6. ↑ Sun, F., Pang, X., Xie, T., Zhai, Y., Wang, G., & Sun, F. (2015). BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli by using BrkA autotransporter. Microbial Cell Factories, 14, 1-12. https://doi.org/10.1186/s12934-015-0316-3