Difference between revisions of "Part:BBa K4722010"
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===Protein Expression===
 
===Protein Expression===
  
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{|
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| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/parts-figure4.jpg></html>
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|-
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| '''Figure 1.''' (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h
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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
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|}
  
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{|
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| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/parts-figure5.jpg></html>
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|-
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| '''Figure 2.'''  (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h
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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
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|}
  
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===Enzyme Activity===
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{|
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TBD
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| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/parts-figure6.jpg></html>
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|-
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| '''Figure 3.'''  (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h
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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
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|}
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{|
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| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/parts-figure7.jpg></html>
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|-
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| '''Figure 4.'''  (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
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===References===
 
===References===

Revision as of 06:29, 9 October 2023

LppOmpA

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

It's A protein, outer membrane protein A, membrane entity.

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 LppOmpA 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 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 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

Figure 4. (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

References