Coding

Part:BBa_K4722018

Designed by: Anqi Chen   Group: iGEM23_BJEA-China   (2023-10-09)

LppOmpA-linker-NicX-Histag

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1241
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1241
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1241
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1241
    Illegal NgoMIV site found at 1400
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

LppOmpA, linker, NicX[1][2], Histag are fused together

Design Consideration

The construction includes: NicX with LppOmpA, Histag and linker NicX was genetically connected with LppOmpA and linker 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. Enzymatic cleavage was performed at the NcoI and XhoI restriction sites, allowing for the precise integration of LppOmpA-linker-NicX-histag. The part contains Histag and does not require Histag on the plasmid.

Protein Expression

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

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
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