Team:RDFZ-China/Basic Parts

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Basic Parts

We are looking for additional promoter that responds to PcauAM. Form literature researching, we found a promoter sequence p3B5B.

1. Organism of p3B5B (BBa_K3395009):

p3B5B is derived from acinetobacter sp. ADP1 [1]

2. Design of p3B5B:

We constructed the promoter into the low copy p15A plasmid with YFP downstream its regulatory protein PcauAM. We transformed pET28b_p3B5B vector into E .coli BL21 for testing.

3. Experimenting（compared to p3B5C）:

PcauAM-YFP: excitation:485nm, emission:525nm:

The data in figure 15 are the tested data of PcaUAM-YFP under emission light 535nm.

When PCA molecular is not added, the fluorescence intensity divided by bacterial concentration of pcauAM-p3b5b and pcauAM-p3b5c are both about 3000. And when PCA is added, the YFP protein expression of the two sensing systems has increased to different degrees, and the value of F/A is increased when reflected in the image. The data shows both p3b5b and p3b5c promoter can be controlled by the pcauAM activator. And under the horizontal comparison between p3b5b and p3b5c promoter, p3b5b promoter have much higher sensitivity than p3b5c promoter.

We are looking for additional tryptophan hydroxylase to catalyze 5-HTP synthesis. From our literature research we found mouse sourced TPH1 for characterization.

1. Origin of musTPH1(BBa_K3395001):

musTPH1 is derived from mouse. 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 [2]

2. Design of musTPH1：

musTPH1 is construct on pET28-b plasmid and bind with upstream LacI sequence. We transformed pET28b_TPH1 vector into E .coli BL21 for expression.

Reference

[1]Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature chemical biology, 15(2), 196-204.

[2]: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.