Coding

Part:BBa_K4722000

Designed by: Chenzhang Ma   Group: iGEM23_BJEA-China   (2023-10-07)
Revision as of 14:40, 8 October 2023 by Chenzhang (Talk | contribs)


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, 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. The origin of this partial information can be attributed to the pioneering work of Jimenez et al., as documented in their study titled 'Gut Bacteria Alleviate Smoking-Related Non-Alcoholic Steatohepatitis (NASH) by Degrading Gut Nicotine.' Additionally, the nic cluster originating from Pseudomonas putida KT2440 has relevance in this context.

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.

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

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Parameters
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