Coding

Part:BBa_K4722011

Designed by: Ziqian Dai   Group: iGEM23_BJEA-China   (2023-10-09)
Revision as of 06:38, 9 October 2023 by Daisy Dai (Talk | contribs)

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INPNC

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 72
    Illegal NgoMIV site found at 405
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Ice nucleating protein (INP) is a protein species identified within the bacterium Xanthomonas campestris pc. campestris BCRC. Its primary function is to catalyze the nucleation and subsequent formation of ice crystals. The structural composition of the INP protein comprises distinct domains: an N-terminal region that exhibits an affinity for interaction with the phospholipid membrane, a C-terminal hydrophilic region that remains exposed to the outer membrane environment, and a central amino acid motif responsible for instigating the ice nucleation capabilities inherent to the INP protein. Intriguingly, while the central amino acid motif crucial for ice nucleation is not essential for sustaining surface display characteristics, it has been subjected to truncation. Consequently, only the N-terminal segment, consisting of 179 amino acids, and the C-terminal segment, comprising 49 amino acids, were retained, yielding a modified protein known as INPNC. Remarkably, INPNC retains its capability for membrane display despite the alterations.


Design Consideration

The genetic construct was ligated into a pET28a plasmid vector and subsequently introduced into Escherichia coli strain BL21 (DE3). NicX was genetically connected with INPNC 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
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
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