Difference between revisions of "Part:BBa K4722001"

 
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===Usage and Biology===
 
  
 
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<partinfo>BBa_K4722001 SequenceAndFeatures</partinfo>
 
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===Functional Parameters===
 
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<partinfo>BBa_K4722001 parameters</partinfo>
 
 
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===Usage and Biology===
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J1 represents a fusion protein known for its multifaceted roles, encompassing the regulation of plasma pH, facilitation of metabolite and fatty acid transport, and serving as a pivotal conduit for drug transportation within the bloodstream<ref>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</ref>. Notably, J1 exhibits the capacity to form a fusion with the nicotine-degrading enzyme NicA2. This union serves the purpose of enhancing the in vivo stability of the NicA2 protein, thereby mitigating the susceptibility of NicA2 to renal filtration.
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It is noteworthy, however, that the mere addition of the J1 fusion protein to either the N- or C-terminus of NicA2 induces profound alterations in both the molecular structure and enzymatic activity of NicA2.
 +
 +
===Design Consideration===
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The genetic construct was ligated into a pET28a plasmid vector and subsequently introduced into Escherichia coli strain BL21 (DE3).
 +
To enhance the stability of NicX and ▲50NicA2 within the human body, the J1 fusion protein was strategically linked in front of them.
 +
Enzymatic cleavage was performed at the NcoI and XhoI restriction sites, allowing for the precise integration of J1-NicX and J1-▲50NicA2. The original His tag on the plasmid was retained, which is useful for subsequent protein purification steps.
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===Protein Expression===
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{|
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| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/parts-figure3.jpg></html>
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|-
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| '''Figure 1.''' (a) SDS-PAGE of J1-▲50NicA2(1458bp)&J1-NicX(1446bp)&J1-NicX(1446bp) &▲50NicA2(1305bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: ▲50NicA2 (1305bp)Supernatant 3: J1-▲50NicA2 (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: ▲50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM IPTG (b) 1: ▲50NicA2 (1305bp)Supernatant 3: 37℃ J1-▲50NicA2 (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℃ ▲50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM IPTG
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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-▲50NicA2 (1458bp) 2: ▲50NicA2 (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-▲50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)Supernatant (b) 1: J1-▲50NicA2 (1458bp) 2: ▲50NicA2 (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-▲50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)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-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
 +
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-▲50NicA2 (1458bp)Washing buffer 12: NicX(1293bp)Washing buffer 13: J1-NicX (1446bp)Washing buffer 14: J1-▲50NicA2 (1458bp)Washing buffer
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|}
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===Enzyme Activity===
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TBD
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===References===

Revision as of 16:50, 8 October 2023

J1

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

J1 represents a fusion protein known for its multifaceted roles, encompassing the regulation of plasma pH, facilitation of metabolite and fatty acid transport, and serving as a pivotal conduit for drug transportation within the bloodstream[1]. Notably, J1 exhibits the capacity to form a fusion with the nicotine-degrading enzyme NicA2. This union serves the purpose of enhancing the in vivo stability of the NicA2 protein, thereby mitigating the susceptibility of NicA2 to renal filtration. It is noteworthy, however, that the mere addition of the J1 fusion protein to either the N- or C-terminus of NicA2 induces profound alterations in both the molecular structure and enzymatic activity of NicA2.

Design Consideration

The genetic construct was ligated into a pET28a plasmid vector and subsequently introduced into Escherichia coli strain BL21 (DE3). To enhance the stability of NicX and ▲50NicA2 within the human body, the J1 fusion protein was strategically linked in front of them. Enzymatic cleavage was performed at the NcoI and XhoI restriction sites, allowing for the precise integration of J1-NicX and J1-▲50NicA2. 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-▲50NicA2(1458bp)&J1-NicX(1446bp)&J1-NicX(1446bp) &▲50NicA2(1305bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: ▲50NicA2 (1305bp)Supernatant 3: J1-▲50NicA2 (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: ▲50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM IPTG (b) 1: ▲50NicA2 (1305bp)Supernatant 3: 37℃ J1-▲50NicA2 (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℃ ▲50NicA2(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-▲50NicA2 (1458bp) 2: ▲50NicA2 (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-▲50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)Supernatant (b) 1: J1-▲50NicA2 (1458bp) 2: ▲50NicA2 (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-▲50NicA2 (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-▲50NicA2 (1458bp)Washing buffer 12: NicX(1293bp)Washing buffer 13: J1-NicX (1446bp)Washing buffer 14: J1-▲50NicA2 (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