Difference between revisions of "Part:BBa K3606035"
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<p>We have successfully cloned NSP4/PhoA/OmpA guided CaAP(Calcium Absorption Peptide) under the regulation of the iPTG inducible promoter, Ptac, and expressed it in E.coli.BL21. As shown in the figure below,PhoA can guide CaAP to secrete into the extracellular space.</p>
 
<p>We have successfully cloned NSP4/PhoA/OmpA guided CaAP(Calcium Absorption Peptide) under the regulation of the iPTG inducible promoter, Ptac, and expressed it in E.coli.BL21. As shown in the figure below,PhoA can guide CaAP to secrete into the extracellular space.</p>
 
[[File:T--Fudan--denoted_gel2_signalpeptides.png|none|400px|thumb|Figure1.NSP4/PhoA/OmpA-CaAP expression with and without iPTG induction in the bacterial lysate.]]
 
[[File:T--Fudan--denoted_gel2_signalpeptides.png|none|400px|thumb|Figure1.NSP4/PhoA/OmpA-CaAP expression with and without iPTG induction in the bacterial lysate.]]
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[[File:T--Fudan--File:T--Fudan--denoted_gel3_signalpeptides.png|none|400px|thumb|Figure2.NSP4/PhoA/OmpA-CaAP expression expression with and without iPTG induction in the culture medium concentrated by TCA.]]
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[[File:T--Fudan--denoted_gel3_signalpeptides.png|none|400px|thumb|Figure2.NSP4/PhoA/OmpA-CaAP expression expression with and without iPTG induction in the culture medium concentrated by TCA.]]
  
 
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Revision as of 01:50, 28 October 2020


PhoA-CaAP

We fused PhoA to the N-terminal of CaAP for its secretion into the extracellular space.

Usage and Biology

The secretion of CaAP(Calcium Absorption Peptide) after its expression is indispensable for it to display the robust function of combining calcium. Here, we decided to achieve CaAP secretion via the type II secretion system, which generally contains two steps. The first step involves some signal peptides targeted to the Sec secretory pathways, which are responsible for transporting CaAP across the inner membrane into the periplasm. For Gram-negative bacteria like E.coli, the second step can then take place once CaAP is in the periplasm, as the intrinsic protein complex GspD creates a pore in the outer cell membrane through which CaAP can be secreted out of the bacteria.

Part BBa_K3606035 is a new composite part designed to allow CaAP secretion from our engineered E.coli strain by adding a signal peptide alkaline phosphatase (PhoA) to the N terminus of CaAP. As a functional peptide that allows surface anchoring of its fused gene product, PhoA enables CaAP to be presented on cellular surface of E.coli.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 138
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

We have successfully cloned NSP4/PhoA/OmpA guided CaAP(Calcium Absorption Peptide) under the regulation of the iPTG inducible promoter, Ptac, and expressed it in E.coli.BL21. As shown in the figure below,PhoA can guide CaAP to secrete into the extracellular space.

Figure1.NSP4/PhoA/OmpA-CaAP expression with and without iPTG induction in the bacterial lysate.
Figure2.NSP4/PhoA/OmpA-CaAP expression expression with and without iPTG induction in the culture medium concentrated by TCA.