Part:BBa_K4907102

I0500-B0034-LMT-his tag-linker-gfp-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.

GFP

GFP is a kind of green fluorescence protein, whose excitation wavelength is 488 nm and emission wavelength is 533 nm. When expressed in engineered bacteria, GFP will emit green fluorescence under light of 488 nm wavelength(1).

Usage and Biology

To test whether LMT signal peptide can guide target proteins secret or not, this basic part BBa_K4907003 was constructed which codes the fused protein LMT-GFP. GFP can be guided into extracellular circumstance by LMT, then the fluorescence can present the success of secretion. Moreover, to guarantee both LMT and GFP can fold normally, we add a linker between them.

Characterization

Agarose gel electrophoresis (AGE)

BBa_I0500, BBa_B0034, BBa_K4907003 and BBa_B0015 were assembled to obtain the composite part BBa_K4907102, which are inserted on the expression vector pSB1C3 by standard assembly. The constructed plasmids are transformed into E. coli DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Fig. 1 Colony PCR of BBa_K4907101_pSB1C3 in E. coli DH10β

Reference

T. D. Craggs, Green fluorescent protein: structure, folding and chromophore maturation. Chem. Soc. Rev. 38, 2865-2875 (2009).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961