Part:BBa_K4907105
I0500-B0034-LMT-his tag-linker-sfibp-B0015
Biology
LMT
LMT is a signal peptide from the lytic murein transglycosylase of Vibrio natriegens which is used to secrete the protein to the periplasm.
SfIBP
SfIBP is a kind of antifreeze proteins (AFPs) found from Shewanella frigidimarina (1). It is capable of preventing liquid from freezing within a certain temperature range and inhibiting ice growth, which is defined as Thermal Hysteresis (TH) and ice recrystallization inhibition (IRI) respectively. Additionally, the IRI activity of SfIBP is much higher than other AFPs.
Usage and Biology
To test whether LMT signal peptide can realize the secretion of SfIBP or not, we constructed this basic part BBa_K4907006 which codes the fused protein LMT-sfibp. If LMT performs a function, SfIBP can be detected in the supernatant by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE). Similarly, a linker was added between LMT and sfibp to retain its function.
Characterization
Agarose gel electrophoresis (AGE)
BBa_I0500, BBa_B0034, BBa_K4907006 and BBa_B0015 was assembled to obtain the composite part BBa_K4907105, which are inserted on the expression vector pSB1C3 by standard assembly. The constructed plasmids are transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.
Reference
- T. D. R. Vance, L. A. Graham, P. L. Davies, An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin. The FEBS J. 285, 1511-1527 (2018).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 1993
Illegal AgeI site found at 2008 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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