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
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.
Background
In order to accomplish this aim, we tested pFliC using a range of concentrations spanning three orders of magnitude. Finally, we evaluated the pFliC’s function using three reporter genes, eCFP, eGFP, and sfGFP to provide a more comprehensive characterization
We used E. coli strain MC1061, as it is the workhorse chassis for our system.
Results
eCFP
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.
eGFP
Fig.5:Adding acetate enabled the expression of eGFP, as pFliC is a SCFA-inducible promoter. We measure emission at 488nm, 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.6: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 acetate seems to prevent cell growth.
Fig.7:. Adding propionate enabled the expression of eGFP, as pFliC is a SCFA-inducible promoter. We measure emission at 488nm, 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.8: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 acetate prevents bacteria to grow.
sfGFP
Fig.9: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.10: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.11: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.12: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.
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
Thessaly 2021 Characterization
Fig.1:Normalized fluorescence of PFlic NOT-GATE. Measurements to test the pFlic module response to Sodium Butyrate. We expected the signal of pFlic under the lowest concentration of sodium butyrate (0,00002mM) to be the highest. Negative control: E.coli BL21 (DE3) Gold with empty vector ω2.
Fig.2:PFlic module, level a, Normalized fluorescence. Measurements to test the pFlic module response to Sodium Butyrate. We expected the signal of pFlic under the highest concentrations of sodium butyrate to be the highest, but that didn’t happen. Negative control (C): E.coli MC1061 empty vector a1R with and without SCFAs.
Fig.3:PFlic module Normalized fluorescence. Measurements to test the pFlic module response to Sodium Acetate. We expected the signal of pFlic under the highest concentrations of sodium butyrate to be the highest, but that didn’t happen. Negative control (C): E.coli MC1061 empty vector a1R with and without SCFAs.
Fig.4:PFlic module, level a, Normalized fluorescence. Measurements to test the pFlic module response to Sodium Butyrate. We expected the signal of pFlic under the highest concentrations of sodium butyrate to be similar to positive control, but that didn’t happen. Negative control: E.coli MC1061 empty vector a1R with and without SCFAs. Positive control: TU with sfGFP only LacO, with and without SCFAs.
Fig.5:PFlic module, Normalized fluorescence. Measurements to test the pFlic module response to Sodium Acetate. We expected the signal of pFlic under the highest concentrations of sodium butyrate to be similar to positive control, which did not happen. Negative control: E.coli MC1061 empty vector a1R with and without SCFAs. Positive control: TU with sfGFP only LacO, with and without SCFAs.
Fig.6: PFlic module, level a, Normalized fluorescence. Measurements to test the pFlic module response to Sodium Butyrate. We expected the signal of pFlic under the highest concentrations of sodium butyrate to be similar to positive control, but that didn’t happen. Negative control: E.coli MC1061 empty vector a1R with and without SCFAs. Positive control: TU with sfGFP only LacO, with and without SCFAs.
Fig.7: PFlic module Normalized fluorescence. Measurements to test the pFlic module response to Sodium Acetate. We expected the signal of pFlic under the highest concentrations of sodium butyrate to be similar to positive control but that didn’t happen. Negative control: E.coli MC1061 empty vector a1R with and without SCFAs. Positive control: TU with sfGFP only LacO, with and without SCFAs.
Conclusion
Our experiments led us to the conclusion that promoter Flic is not induced by sodium acetate or sodium butyrate, regardless of their concentrations, under these experimental conditions. At fig.1, the signal of pFlic under the lowest concentration of sodium butyrate was expected to be the highest. Additionally, the signal of pFlic (fig.2,3,4,5,6,7) under the highest concentrations of sodium butyrate and sodium acetate should have been the highest but that did not happen.
