Difference between revisions of "Part:BBa K4722011"

Latest revision as of 06:38, 9 October 2023

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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 __NOTOC__
 <partinfo>BBa_K4722011 short</partinfo>
-<!-- Add more about the biology of this part here
-===Usage and Biology===
 <!-- -->
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 <partinfo>BBa_K4722011 SequenceAndFeatures</partinfo>
-<!-- Uncomment this to enable Functional Parameter display
-===Functional Parameters===
-<partinfo>BBa_K4722011 parameters</partinfo>
 <!-- -->
+===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===
+{|
+| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/parts-figure1.jpg></html>
+|-
+| '''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
+|}
+{|
+| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/parts-figure2.jpg></html>
+|-
+| '''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
+|}