Difference between revisions of "Part:BBa K4722005"
 
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The genetic construct was ligated into a pET28a plasmid vector and subsequently introduced into Escherichia coli strain BL21 (DE3).
 
The genetic construct was ligated into a pET28a plasmid vector and subsequently introduced into Escherichia coli strain BL21 (DE3).
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Enzymatic cleavage was performed at the NcoI and XhoI restriction sites, allowing for the precise integration of NicX-V16G. The original His tag on the plasmid was retained, which is useful for subsequent protein purification steps.
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Enzymatic cleavage was performed at the NcoI and XhoI restriction sites, allowing for the precise integration of NicX-L48Q. The original His tag on the plasmid was retained, which is useful for subsequent protein purification steps.
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A point mutation was introduced to NicX, altering its gene sequence by replacing the 16th amino acid from V to G, with the aim of potentially enhancing its enzyme activity.
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A point mutation was introduced to NicX, altering its gene sequence by replacing the 48th amino acid from L to Q, with the aim of potentially enhancing its enzyme activity.
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===Protein Expression===
 
===Protein Expression===

Latest revision as of 17:26, 8 October 2023

NicX-L48Q

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). The 48th amino acid in the NicX peptide chain changes from L to Q.

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-L48Q. The original His tag on the plasmid was retained, which is useful for subsequent protein purification steps. A point mutation was introduced to NicX, altering its gene sequence by replacing the 48th amino acid from L to Q, with the aim of potentially enhancing its enzyme activity.


Protein Expression

Figure 1. (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

Enzyme Activity

TBD

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