Team:RDFZ-China/Proof Of Concept

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Proof of concept

To prove that our idea is practical, we have broken our circuit down into two components and tested them individually. For sensors, we linked GFP or YFP reportor to the downstream of the promoters of our three sensor candidates. We transformed each to E.coli BL21 and processed it with a gradient of PCA to determine each's sensitivity against PCA. From this practice, we found out that PcaUAM activator combine with p3B5B promoter meets our expectations to work as a sensor.

Through the data we got from fluorescence signal testing. PcaU-pPca and PcauAM-p3b5b showed significant response against pure PCA. When PCA is added, the two systems present high fluorescent intensity. Specifically, in PcaUAM sensing system, p3B5B promoter has the lowest leakage at 0 PCA. Hence, PcaUAM-p3B5B has been decided to fulfill our expectation for a sensitive, yet leakage preventing sensor for our project.

For our effector, we have characterized human-sourced TPH1 with lac operon and induced for expression. Then we conducted enzyme activity assay and proved our enzyme is competent for our main goal.

We also collected a mouse-sourced musTPH1 and plan for the same characterization and testing. It is known to catalyze the same reaction human TPH1 did. Although the full experimentation data haven’t been complete on this biobrick, musTPH1 is proven to have high expression in the bacteria strain E.coli MC1061 with proper activity to produce concentration greater than 1mM under certain conditions by the article Park D H, Stone D M, Kim K S, et al. Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli[J]. Molecular and Cellular Neuroscience, 1994, 5(1): 87-93.

By the enzymatic activity assay, we can show 5-HTP concentration varied by time and different concentrations of enzymes. We build the model of TPH1 enzyme activity which will be explained in the model page. The data shows our TPH1 enzyme functions very well when tryptophan and other catalases are present is the solution. Hence, we can combine our TPH1 system to the previous sensor, and consequently get a complete TPH1-generating system to achieve our goal, which is to synthesize 5-HTP in gut using PCA from tea.

For further experimentation next year, we plan to first apply our circuit to E.coli Nissle 1917, as this is the strain we hope to use in practice. We also hope to test the sensor unit with urine hours after tea intake, as it is a good reference of the concentration of PCA existing in intestine after drinking tea and thus primarily prove our design works in human body for PCA sensing. We also will want to combine the two components with the negative feedback loop and test it as an integrated pathway in terms of PCA sensitivity and TPH1 expression. For PCA sensitivity we will use human urine as well as pure PCA gradient as testing agent. We will have GFP linked downstream to TPH1 and thus determine TPH1 expression. Another step further, we hope to test the full design in a virtual intestinal environment, to prove it works as a whole in its supposed environment as well.

Reference

[1] Park D H, Stone D M, Kim K S, et al. Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli[J]. Molecular and Cellular Neuroscience, 1994, 5(1): 87-93.

[2] P. G. Pietta, P. Simonetti, C. Gardana, A. Brusamolino, P. Morazzoni, E. Bombardelli, Catechin metabolites after intake of green tea infusions. Biofactors 8, 111–118 (1998).