Part:BBa_K3505028

PfliC:RBS-eCFP-terminator

eCFP BBa_K3505019uder control of a SCFAs inducible promoter pFLliC BBa_K2924016.

Fig.1:pFliC-eCFP-Terminator

Usage and Biology

This Trancriscription Unit (TU) is activated form SCFAs and more specifically from Butyrate. Exressing the eCFP protein for report gene, representing the activity of the promoter FliC.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in BBa_K3505008 and has overhangs compatible for Golden Braid cloning.

Verification of cloning

Fig.2:(U=Uncut C=Cut) Restriction Enzyme with PvuII, Expected bands in bp 3016 + 816

Experimental Use and Experinece

Fig.1: Adding acetate enabled the expression of eCFP, as pFliC is a SCFA-inducible promoter. We measure the absorbance at 434nm , adding different concentrations of acetate, 0,02mM, 0,2mM,2mM,20mM and 200mM. Our time-points were 0,4,8 and 20 hours, while we incubated each culture at 37oC and 210 rpm. The result of the absorbance at 434nm is divided by cell growth (600nm), in order to normalize all values.

Fig.2: Adding propionate enabled the expression of eCFP, as pFliC is a SCFA-inducible promoter. We measure the absorbance at 434nm , adding different concentrations of propionate, 0,02mM, 0,2mM,2mM,20mM and 200mM. Our time-points were 0,4,8 and 20 hours, while we incubated each culture at 37oC and 210 rpm. The result of the absorbance at 434nm is divided by cell growth (600nm), in order to normalize all values.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]