Part:BBa_K4907007

LMT-his tag-linker-tmafp

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.

TmAFP

TmAFP, found in Tenebrio molitor, is the one of antifreeze proteins (AFPs) (1). It is capable of preventing liquid freezing and inhibiting ice growth, which is defined as Thermal Hysteresis (TH) and ice recrystallization inhibition (IRI) respectively. Notably, TmAFP possesses the highest TH activity between all AFPs that have been found.

Usage and Biology

To test whether the LMT signal peptide can realize the secretion of TmAFP or not, this basic part BBa_K4907006 was constructed which codes the fused protein LMT-tmafp. If LMT performs a function, TmAFP can be detected in supernatant by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE). Similarly, a linker was added between LMT and TmAFP to retain its function.

Characterization

Agarose gel electrophoresis (AGE)

BBa_I0500, BBa_B0034, BBa_K4907007 and <parinfo>BBa_B0015</partinfo> was assembled to obtain the composite part BBa_K4907106, which were inserted on the expression vector pSB1C3 by standard assembly. The constructed plasmids were 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_K4907106_pSB1C3 in E. coli BL21(DE3)

Reference

1. U. S. Midya, S. Bandyopadhyay, Elucidating the Sluggish Water Dynamics at the Ice-Binding Surface of the Hyperactive Tenebrio molitor Antifreeze Protein. J. Phys. Chem. B 127, 121-132 (2023).

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 325
  
• 1000
  COMPATIBLE WITH RFC[1000]