Composite

Part:BBa_K3505028

Designed by: Venetios Michelioudakis, Magdalini Koroxenidou   Group: iGEM20_Thessaly   (2020-10-20)
Revision as of 20:56, 27 October 2020 by Asteriat (Talk | contribs) (→‎Design Notes)


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 GoldenBraid 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 emission at 475nm, adding different concentrations of acetate. Our time-points were 0, 4, 8, and 20 hours, while we incubated each culture at 37oC and 210 rpm.
Fig.2:Different concentrations and time-points (0, 4, 8 and 20 hours) of acetate affect cell growth. As time passes there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding 20mM and 200mM acetate seems to prevent cell growth.
Fig.3:. Adding propionate enabled the expression of eCFP, as pFliC is a SCFA-inducible promoter. We measure emission at 475nm, adding increasing concentrations of propionate.. Our time-points were 0, 4, 8 and 20 hours, while we incubated each culture at 37oC and 210 rpm.
Fig.4:Different concentrations and time-points (0, 4, 8 , 20 hours) of acetate affect cell growth. As time passes there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding propionate seems to affect cell growth.

Conclusion

The graphs above indicate that adding acetate and propionate can provoke the expression of the reporter genes. However, based on the concentration and the time-point that the measurement is taken, the results change. As time passes, the expression of each fluorescent protein is higher. Furthermore, there is the maximum expression of these three reporter genes when we add 2mM acetate or propionate accordingly, while it is indicated that adding increasing (20mM, 200mM) concentration of acids and, especially, acetate prevents cell growth. That may occur, due to their toxicity to the cells which may lead to unsettling results.

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]
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Categories
Parameters
None