Difference between revisions of "Part:BBa K3896008"
 
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We selected the PmrA-PmrB two-component system as a vector for the core binding domain of hAPN. This system, present in Salmonella and most strains, is initially an Fe(III)-sensitive regulatory system[1] that contains a histidine kinase (HK) on its transmembrane portion, PmrB, for sensing specific environmental stimuli, and a corresponding response regulator (RR), PmrA, for mediating cellular responses.
 
We selected the PmrA-PmrB two-component system as a vector for the core binding domain of hAPN. This system, present in Salmonella and most strains, is initially an Fe(III)-sensitive regulatory system[1] that contains a histidine kinase (HK) on its transmembrane portion, PmrB, for sensing specific environmental stimuli, and a corresponding response regulator (RR), PmrA, for mediating cellular responses.
  
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The target of the assay is the S protein, a monomer of the echinoderm glycoprotein on the HCoV-229E envelope, responsible for binding to human aminopeptidase N (hAPN) [2]. By reviewing the data, we found that in the complex protein of hAPN and S protein, the core binding domain of hAPN is concentrated between Ala281 and Gly330, while the RBD of S protein is Lys201 to Ser321 (Fig.1). Therefore, we intercepted the peptide sequence of the hAPN fragment as our core detection binding domain.
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The target of the assay is the S protein, a monomer of the echinoderm glycoprotein on the 229E-CoV envelope, responsible for binding to human aminopeptidase N (hAPN) [2]. By reviewing the data, we found that in the complex protein of hAPN and S protein, the core binding domain of hAPN is concentrated between Ala281 and Gly330, while the RBD of S protein is Lys201 to Ser321 (Fig.2). Therefore, we intercepted the peptide sequence of the hAPN fragment as our core detection binding domain.
+
  
  
 
https://static.igem.org/mediawiki/parts/0/0a/T--NEU_CHINA--hapn_binding_domain_suoxiaoban.png
 
https://static.igem.org/mediawiki/parts/0/0a/T--NEU_CHINA--hapn_binding_domain_suoxiaoban.png
  
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'''Fig.2 Binding domain of S protein and hAPN'''
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'''Fig.1 Binding domain of S protein and hAPN'''
  
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We replaced the Fe (III) sensitive domains Trp34 to Glu64 of the original PmrB with the core binding domain of hAPN protein to accept the stimulation of S protein of 229E-CoV. After detecting the S protein of 229E-CoV, the sensor kinase PmrB autophosphorylates the highly conserved histidine residue, and then transfers the phosphate group to the conserved aspartate residue in its homologous reaction regulator PmrA. Then phosphorylated PmrA protein combined with promoter PmrC sequence to activate the expression of reporter gene.
+
We replaced the Fe (III) sensitive domains Trp34 to Glu64 of the original PmrB with the core binding domain of hAPN protein to accept the stimulation of S protein of HCoV-229E . After detecting the S protein of HCoV-229E,the sensor kinase PmrB autophosphorylates the highly conserved histidine residue, and then transfers the phosphate group to the conserved aspartate residue in its homologous reaction regulator PmrA. Then phosphorylated PmrA protein combined with promoter PmrC sequence to activate the expression of reporter gene.
  
 
In order to verify the detection function of our engineered bacteria, we designed a protein characterization experiment. In the experiment, we added 1mM IPTG to induce the expression of PmrB and PmrA, and added extracted S protein after 2h of induced expression.When the engineered bacteria received the stimulation of S protein, the transmembrane protein PmrB containing a histidine kinase (HK) underwent autophosphorylation and subsequently transferred the phosphate group to the conserved aspartate residue in the intracellular regulator PmrA, making PmrA to activate transcription of the PmrC promoter and express the downstream reporter gene EGFP.
 
In order to verify the detection function of our engineered bacteria, we designed a protein characterization experiment. In the experiment, we added 1mM IPTG to induce the expression of PmrB and PmrA, and added extracted S protein after 2h of induced expression.When the engineered bacteria received the stimulation of S protein, the transmembrane protein PmrB containing a histidine kinase (HK) underwent autophosphorylation and subsequently transferred the phosphate group to the conserved aspartate residue in the intracellular regulator PmrA, making PmrA to activate transcription of the PmrC promoter and express the downstream reporter gene EGFP.
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We transferred the plasmid into <i>E.coli BL21(DE3)</i> and set up the following experimental groups, which yielded results consistent with expectations.
 
We transferred the plasmid into <i>E.coli BL21(DE3)</i> and set up the following experimental groups, which yielded results consistent with expectations.
  
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figure(3 groups)
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We also set up different concentration gradients and tested the fluorescence intensity and absorbance of the engineered bacterial solution, and processed the experimental data to arrive at the optimum concentration(Fig.2).
  
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We also set up different concentration gradients and tested the fluorescence intensity and absorbance of the engineered bacterial solution, and processed the experimental data to arrive at the optimum concentration.
+
https://static.igem.org/mediawiki/parts/8/84/T--NEU_CHINA--pmrc_hapn_suoxiaoban.png
  
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figure(result)
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'''Fig.2 Fluorescence intensity of EGFP.Ctrl:Detection bacterial.IPTG:Detection bacterial+IPTG.IPTG+S Pr:Detection bacterial+IPTG+S protein'''
  
 
A series of experimental results demonstrate the feasibility of the PmrCAB system for MERS-CoV detection.
 
A series of experimental results demonstrate the feasibility of the PmrCAB system for MERS-CoV detection.

Latest revision as of 16:19, 20 October 2021


The gene of recombinant PmrCAB (HAPN) two-component system.

We selected the PmrA-PmrB two-component system as a vector for the core binding domain of hAPN. This system, present in Salmonella and most strains, is initially an Fe(III)-sensitive regulatory system[1] that contains a histidine kinase (HK) on its transmembrane portion, PmrB, for sensing specific environmental stimuli, and a corresponding response regulator (RR), PmrA, for mediating cellular responses.

The target of the assay is the S protein, a monomer of the echinoderm glycoprotein on the HCoV-229E envelope, responsible for binding to human aminopeptidase N (hAPN) [2]. By reviewing the data, we found that in the complex protein of hAPN and S protein, the core binding domain of hAPN is concentrated between Ala281 and Gly330, while the RBD of S protein is Lys201 to Ser321 (Fig.1). Therefore, we intercepted the peptide sequence of the hAPN fragment as our core detection binding domain.


T--NEU_CHINA--hapn_binding_domain_suoxiaoban.png

Fig.1 Binding domain of S protein and hAPN

We replaced the Fe (III) sensitive domains Trp34 to Glu64 of the original PmrB with the core binding domain of hAPN protein to accept the stimulation of S protein of HCoV-229E . After detecting the S protein of HCoV-229E,the sensor kinase PmrB autophosphorylates the highly conserved histidine residue, and then transfers the phosphate group to the conserved aspartate residue in its homologous reaction regulator PmrA. Then phosphorylated PmrA protein combined with promoter PmrC sequence to activate the expression of reporter gene.

In order to verify the detection function of our engineered bacteria, we designed a protein characterization experiment. In the experiment, we added 1mM IPTG to induce the expression of PmrB and PmrA, and added extracted S protein after 2h of induced expression.When the engineered bacteria received the stimulation of S protein, the transmembrane protein PmrB containing a histidine kinase (HK) underwent autophosphorylation and subsequently transferred the phosphate group to the conserved aspartate residue in the intracellular regulator PmrA, making PmrA to activate transcription of the PmrC promoter and express the downstream reporter gene EGFP.

We transferred the plasmid into E.coli BL21(DE3) and set up the following experimental groups, which yielded results consistent with expectations.

We also set up different concentration gradients and tested the fluorescence intensity and absorbance of the engineered bacterial solution, and processed the experimental data to arrive at the optimum concentration(Fig.2).

T--NEU_CHINA--pmrc_hapn_suoxiaoban.png

Fig.2 Fluorescence intensity of EGFP.Ctrl:Detection bacterial.IPTG:Detection bacterial+IPTG.IPTG+S Pr:Detection bacterial+IPTG+S protein

A series of experimental results demonstrate the feasibility of the PmrCAB system for MERS-CoV detection.

Reference

[1].Shangwen, Zhang , et al. "Fabrication and Characterization of One Interpenetrating Network Hydrogel Based on Sodium Alginate and Polyvinyl Alcohol." Journal of Wuhan University of Technology-Mater. Sci. Ed. 34.003(2019):744-751.

[2] Li Z, Tomlinson AC, Wong AH, Zhou D, Desforges M, Talbot PJ, Benlekbir S, Rubinstein JL, Rini JM. The human coronavirus HCoV-229E S-protein structure and receptor binding. Elife. 2019 Oct 25;8:e51230. 

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 96
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2343
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1081
  • 1000
    COMPATIBLE WITH RFC[1000]