Part:BBa_K4907006

LMT-his tag-linker-sfibp

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.

Fig. 1 Colony PCR of BBa_K4907105_pSB1C3 in E. coli BL21(DE3)

Reference

1. 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

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 757
  Illegal AgeI site found at 772
  
• 1000
  COMPATIBLE WITH RFC[1000]