Regulatory

Part:BBa_K2924016

Designed by: Mirko Kraus   Group: iGEM19_Duesseldorf   (2019-10-14)
Revision as of 13:57, 23 October 2020 by Venetios (Talk | contribs) (→‎Aim)


Promoter fliC from the Escherichia coli genome

Short-chain fatty acid sensitive promoter FliC

Usage and Biology

The promoter fliC was published as a sensitive promoter for short-chain fatty acids, especially for butyrate (C4:0). This promoter was isolated from the Escherichia coli wild type genome. In the wild type the short-chain fatty acids have an impact on the flagellar expression. The PfliC is repressed by leucine-responsive regulatory protein (Lrp). Butyrate can enhance the expression of the flagellar expression like leucine which is a ligand of Lrp. Difference between thus enhancers is that the promoter fliC is only sensitive for the butyrate and not for the leucine 1

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]


Characterization

The promoter was tested for the sensitivity to butyric acid in the culture medium by combining the promoter to an eYFP (BBa_E0030) 2 as a reporter gene in the composite part BBa_K2924017. The concentrations of butyric acid were from 0.5 mM to 20 mM.

Fig.1: Response of PfliC+eYFP (red) to different chain lengths of fatty acids compared to an empty vector control (black). The fluorescence was measured at an excitation wavelength from 497 nm and an emission wavelength from 540 nm.


The experiment showed that the fluorescence doesn't grow with higher concentrations of butyric acid. Surprisingly the fluorescence from the empty vector control rises with higher concentrations while the PfliC shows a falling tendency.



















Thessaly 2020's Characterization

The sensitivity of pFliC in the other SCFAs: Acetate and Propionate

Background

Aim

The Monitoring System of Amalthea comprises three separate modules. We chose to extensively characterize the Prom Module before working with it for the proof of concept. Briefly, the Prom Module is a NOT-GATE that is activated due to the absence of Short-Chain Fatty-Acids (SCFAs). Its key element is a SCFA-inducible promoter, pFliC, which is mainly activated by butyrate. While researching its properties, we realized that it was characterized for its sensitivity to butyric acid, only in a limited way.

Results

References

  • [1]: Toru Tobe,* Noriko Nakanishi, and Nakaba Sugimoto “Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic Escherichia coli” INFECTION AND IMMUNITY, Mar. 2011, p. 1016–1024
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