Difference between revisions of "Part:BBa K3606038"
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===Characterization=== | ===Characterization=== | ||
| − | <p>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 | + | <p>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.</p> |
| − | [[File:T--Fudan-- | + | [[File:T--Fudan--denoted_gel2_OmpA_MBP.png|none|200px|thumb|Figure1.OmpA-MBP expression with and without iPTG induction in the bacterial lysate.]] |
| − | [[File:T--Fudan--gel3_OmpA_MBP.png|none| | + | [[File:T--Fudan--gel3_OmpA_MBP.png|none|200px|thumb|Figure2.OmpA-MBP expression with and without iPTG induction in the culture medium concentrated by TCA.]] |
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Latest revision as of 03:29, 28 October 2020
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1233
Illegal BglII site found at 1732 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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.
