Coding

Part:BBa_K4722014

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

J1-Δ50NicA2

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1335
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

To generate Δ50-NicA2, the initial 50 amino acids at the N-terminus of NicA2 were excised. Subsequently, residues 51 (Glycine) and 52 (Glycine) were linked to ABD035, serving as a native linker, yielding the construct ABD035-Δ50-Nica2, denoted as NicA2-J1. Notably, the deletion of the first 50 N-terminal amino acids and the fusion with the J1 fusion protein did not exert any discernible impact on the enzymatic kinetics[1]. The resultant construct continued to manifest a high affinity for both human and rat albumin, while also demonstrating effectiveness in mitigating somatic manifestations of nicotine withdrawal[2].

Design Consideration

The construction includes: Δ50NicA2 with J1 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 J1-Δ50NicA2. The original His tag on the plasmid was retained, which is useful for subsequent protein purification steps.


Protein Expression

Figure 1. (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 2. (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 3. (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

Enzyme Activity

TBD

References

  1. ↑ 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
  2. ↑ Tang, H., Wang, L., Wang, W., Yu, H., Zhang, K., Yao, Y., & Xu, P. (2013). Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas. PLoS genetics, 9(10), e1003923. https://doi.org/10.1371/journal.pgen.1003923
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