Difference between revisions of "Part:BBa K2871001"

 
 
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A Translocon is a complex of proteins that assemble a pore-like structure in membranes. EspD is part of a group of proteins used for assembly of a translocon that works like a docking site for the bacterial type-3-secretion system (T3SS). This docking site is also the site of protein injection by the T3SS.
 
A Translocon is a complex of proteins that assemble a pore-like structure in membranes. EspD is part of a group of proteins used for assembly of a translocon that works like a docking site for the bacterial type-3-secretion system (T3SS). This docking site is also the site of protein injection by the T3SS.
  
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===Usage and Biology===
 
===Usage and Biology===
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'''Gene expression and Protein purification'''
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EspD was PCR from SIEC E. coli strain and insert into IPTG inducible BBa_K731500 vector under pTac promoter and BBa_B0034 ribosome binding site by USER cloning. The expression vector map is shown in Figure 1. The expression vector is transformed into E. coli and the expression is induced with 1mM IPTG in LB broth. The cell was lysed by sonication and centrifugation. Western blot experiment shows that His-tagged EspD protein is only present in the pellet part of the lysed cell as shown in Figure 2. However, the amount of EspD protein is very low, which prohibit purification.
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[[Image:EspD_vector.jpeg|400px|thumb|Figure 1. '''EspD expression vector map.]]
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[[Image:EspD WB.png|400px|thumb|Figure 2. '''His-tagged EspD protein.]]
  
 
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Latest revision as of 23:16, 17 October 2018


EspD Translocon protein. Membrane protein for T3SS docking.

A Translocon is a complex of proteins that assemble a pore-like structure in membranes. EspD is part of a group of proteins used for assembly of a translocon that works like a docking site for the bacterial type-3-secretion system (T3SS). This docking site is also the site of protein injection by the T3SS.

Usage and Biology

Gene expression and Protein purification

EspD was PCR from SIEC E. coli strain and insert into IPTG inducible BBa_K731500 vector under pTac promoter and BBa_B0034 ribosome binding site by USER cloning. The expression vector map is shown in Figure 1. The expression vector is transformed into E. coli and the expression is induced with 1mM IPTG in LB broth. The cell was lysed by sonication and centrifugation. Western blot experiment shows that His-tagged EspD protein is only present in the pellet part of the lysed cell as shown in Figure 2. However, the amount of EspD protein is very low, which prohibit purification.

Figure 1. EspD expression vector map.
Figure 2. His-tagged EspD protein.

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 422
    Illegal XhoI site found at 1045
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 739
  • 1000
    COMPATIBLE WITH RFC[1000]