Composite

Part:BBa_K3606038

Designed by: Zhenru Zhang   Group: iGEM20_Fudan   (2020-10-23)


PlacIq_lacI_Ptac driven OmpA-MBP

OmpA_MBP is constructed for the test and verification of secretion led by OmpA.

Usage and Biology

MBP(Maltose Binding Protein) is a protein that normally expressed in the cytoplasm of E.coli.

To test the efficiency of our 5 signal peptides for the secretion of the target protein, we fused our 5 signal peptides to MBP to compare their secretion capabilities. The signal peptides and corresponding serial numbers are listed below, through which the biology and characterization of each peptide are available.

Signal Peptide Number for Registry
NSP4 BBa_K3606042
OmpA BBa_K3606043
DsbA BBa_K3606030
PelB BBa_K208004
PhoA BBa_K808028

Moreover, we put the expression of secretion peptide guided MBP under the regulation of Ptac, an iPTG inducible promoter, to achieve better qualification of our secretion peptides.

To test the secretion efficiency of our 5 secretion peptides and compare their secretion capabilities, we also fused them to MBP(Maltose Binding Protein) that normally expressed in the cytoplasm of E.coli. cells. Driven by an iPTG inducible promoter Ptac, we can achieve quantified characterization of these secretion peptides.

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 1233
    Illegal BglII site found at 1732
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1430

Characterization

We have successfully cloned OmpA guided MBP(Maltose Binding Protein) and expressed it in E.coli.BL21. As shown in Figure.1 ,the amount of MBP in the lysate shows no difference with or without iPTG induction, suggesting that OmpA has the strong ability to secrete MBP into the extracellular space.As for the relatively low amount of MBP in the culture medium concentrated by TCA than in the cell lysate(shown in Figure.2), it may be caused by other interference factors during the preparation of protein samples.

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


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