Composite

Part:BBa_K3505030

Designed by: Venetios Michelioudakis, Magdalini Koroxenidou   Group: iGEM20_Thessaly   (2020-10-20)


PfliC:RBS- sfGFP-terminator

sfGCFP BBa_K3505018uder control of a SCFAs inducible promoter pFLliC BBa_K2924016.

Fig.1:pFliC-sfGFP-Terminator

Usage and Biology

This Trancriscription Unit (TU) is activated form SCFAs and more specifically from Butyrate. Exressing the sfGFP 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. U1,C1 pFliC-sfGFP-Terminator. Digested with PvuII. Expecting Bands 3016, 816

Experimental Use and Experinece

Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates

Fig.3:Adding acetate enabled the expression of sfGFP, as pFliC is a SCFA-inducible promoter. We measure emission at 485nm, adding increasing concentrations of acetate. 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 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.5:Adding propionate enabled the expression of sfGFP, as pFliC is a SCFA-inducible promoter. We measure emission at 485nm, 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.6:Different concentrations and time-points of acetate affect cell growth. As time passes (0, 4, 8, and 20 hours) there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding 200mM propionate seems to decrease 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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 246
  • 1000
    COMPATIBLE WITH RFC[1000]
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