Part:BBa_K4907100

I0500-B0034-gfp-B0015

Biology

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 the two signal peptides LMT and KpSP whether can secret target proteins or not, this composite part was constructed to compare the fluorescence intensity.

Characterization

Agarose gel electrophoresis (AGE)

BBa_I0500, BBa_B0034, BBa_K4907036 and BBa_B0015 were assembled to obtain the composite part BBa_K4907100, which are assembled 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_K4907100_pSB1C3 in E. coli DH10β

Fluorescence Intensity Ratio

The plasmid verified by sequencing was successfully transformed into E. coli DH10βThis engineered bacteria with iE.coli</i> DH10β harboring BBa_K4907101_pSB1C3 and iE.coli</i> DH10β were cultivated and induced by 0.2% L-arabinose at 37 ℃ 12 hours. Then 1 mL bacterial liquid of each group was centrifuged, and the fluorescence intensity of the supernatant and the bacterial liquid was measured. As Fig. 2 shows, the fluorescence intensity of supernatant versus bacterial liquid of E. coli DH10βharboring BBa_K4907101_pSB1C3 was lower than the bacteria which express GFP extracellularly.

Fig. 2 Comparation of the fluorescence intensity ratio (supernatant to bacterial culture) between I0500 set as control group.

Reference

1. 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