Difference between revisions of "Part:BBa K4601007"

 
 
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<partinfo>BBa_K4601007 short</partinfo>
 
<partinfo>BBa_K4601007 short</partinfo>
  
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This part is a fusion protein between the Jaws halorhodopsin ([[Part:BBa_K4601002|BBa_K4601002]]), the ''Natronomonas pharaonis'' HtrII transmembrane domain and the ''E. coli'' EnvZ histidine kinase domain.
  
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===Usage and Biology===
 
===Usage and Biology===
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Halorhodopsins are chloride inward pumps activated by light [1]. To detect this activity in ''E. coli'' we designed a halorhodopsin screening system that uses fusion proteins composed of the halorhodopsin of interest, the NpHtrII transmembrane domain and the ''E. coli'' EnvZ histidine kinase domain. This design is based on several considerations:
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-> The HR is covalently linked to the NpHtrII transmembrane domain (via a short linker sequence) to ensure their colocalization
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-> The natural histidine kinase domain of the NpHtrII was removed as it does not specifically interact with an E. coli signaling cascade. It was replaced by an endogenous ''E. coli'' histidine kinase domain.
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-> The ''E. coli'' EnvZ was chosen as a histidine kinase domain as it is part of the widely known EnvZ-OmpR two component system [2].
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In this system (Figure 1), it is expected that in the presence of light the halorhodopsin activation is transmitted via protein-protein interaction to the NpHtrII transmembrane domain, which in turn leads to the autophosphorylation of the EnvZ histidine kinase domain and subsequently the phosphorylation of the OmpR transcription factor. The phosphorylated OmpR dimerises and binds to specific sequences present in the promoter regions of the ompC and ompF genes thus upregulating their expression.
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Based on this property, we designed several reporters in which the pOmpC promoter controls the expression of sfGFP ([[Part:BBa_K4601223|BBa_K4601223]]), LacZ𝛼 (([[Part:BBa_K4601233|BBa_K4601233]]), the ampicillin resistance gene AmpR ([[Part:BBa_K4601243|BBa_K4601243]]) or chloramphenicol resistance gene CmR ([[Part:BBa_K4601253|BBa_K4601253]]).
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Opsin activation should lead to the expression of the reporter gene and the detection of either a fluorescent output, a colorimetric one, or the capacity to grow in the presence of ampicillin or chloramphenicol. The latter systems would constitute a good selection tool for the identification of halorhodopsins with improved properties in different conditions of light.
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To be functional in ''E. coli'' this system requires the knockout of the natural ''envZ' gene.
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<img src="https://static.igem.wiki/teams/4601/wiki/parts-registry/envz-pompr-system.png" width="500px">
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</html>
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Figure 1: Design of the halorhodopsin activity detection system based on fusion proteins and the ''E. coli'' EnvZ/OmpR two component system.
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For the characterisation of this part an expression cassette was assembled and the results are presented on its page in Parts Registry: [[Part:BBa_K4601207|BBa_K4601207]].
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===References===
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[1] Engelhard C, Chizhov I, Siebert F, Engelhard M. Microbial halorhodopsins: light-driven chloride pumps. Chemical Reviews (2018) 118: 10629–10645.
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[2] Mizuno T, Mizushima S. Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes. Molecular Microbiology (1990) 4: 1077–1082.
  
 
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Latest revision as of 19:01, 11 October 2023


Jaws-NpHtrII-EnvZ

This part is a fusion protein between the Jaws halorhodopsin (BBa_K4601002), the Natronomonas pharaonis HtrII transmembrane domain and the E. coli EnvZ histidine kinase domain.

Usage and Biology

Halorhodopsins are chloride inward pumps activated by light [1]. To detect this activity in E. coli we designed a halorhodopsin screening system that uses fusion proteins composed of the halorhodopsin of interest, the NpHtrII transmembrane domain and the E. coli EnvZ histidine kinase domain. This design is based on several considerations:

-> The HR is covalently linked to the NpHtrII transmembrane domain (via a short linker sequence) to ensure their colocalization

-> The natural histidine kinase domain of the NpHtrII was removed as it does not specifically interact with an E. coli signaling cascade. It was replaced by an endogenous E. coli histidine kinase domain.

-> The E. coli EnvZ was chosen as a histidine kinase domain as it is part of the widely known EnvZ-OmpR two component system [2].

In this system (Figure 1), it is expected that in the presence of light the halorhodopsin activation is transmitted via protein-protein interaction to the NpHtrII transmembrane domain, which in turn leads to the autophosphorylation of the EnvZ histidine kinase domain and subsequently the phosphorylation of the OmpR transcription factor. The phosphorylated OmpR dimerises and binds to specific sequences present in the promoter regions of the ompC and ompF genes thus upregulating their expression.

Based on this property, we designed several reporters in which the pOmpC promoter controls the expression of sfGFP (BBa_K4601223), LacZ𝛼 ((BBa_K4601233), the ampicillin resistance gene AmpR (BBa_K4601243) or chloramphenicol resistance gene CmR (BBa_K4601253).

Opsin activation should lead to the expression of the reporter gene and the detection of either a fluorescent output, a colorimetric one, or the capacity to grow in the presence of ampicillin or chloramphenicol. The latter systems would constitute a good selection tool for the identification of halorhodopsins with improved properties in different conditions of light.

To be functional in E. coli this system requires the knockout of the natural envZ' gene.

Figure 1: Design of the halorhodopsin activity detection system based on fusion proteins and the E. coli EnvZ/OmpR two component system.

For the characterisation of this part an expression cassette was assembled and the results are presented on its page in Parts Registry: BBa_K4601207.

References

[1] Engelhard C, Chizhov I, Siebert F, Engelhard M. Microbial halorhodopsins: light-driven chloride pumps. Chemical Reviews (2018) 118: 10629–10645.

[2] Mizuno T, Mizushima S. Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes. Molecular Microbiology (1990) 4: 1077–1082.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 581
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 816
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 957