Design

Overview

In our project, we plan to realize the efficiently production of tryptophan in E.coli. Based on the biosynthesis pathway of tryptophan, aroG(encoding 3-deoxy-7-phosphoheptulonate synthase) and trpBA (encoding tryptophan synthase) were used to improve the yield of tryptophan (Fig.1.1). aroG can divert the intermediate products of glycolysis into the chorismate synthesis pathway, while trpBA can synthesize the precursor chorismate into tryptophan.

We consider that excessive tryptophan production could interfere the proliferation of E.coli, for over-expressed aroG will competitive inhibit the glycolysis pathway while large amount of ATP and NADPH are consumed during the synthesis. Therefore, a toggle-switch circuit are used to reconciling the contradiction between cell proliferation and tryptophan production. In one of the bistable state, over expression of pykA(encoding pyruvate kinase II) is used to eliminate the competitive inhibition of aroG, reducing the production process and enabling a rapid cell proliferation. When cells reach a high density, they can be induced into another state where aroG and trpBA are expressed to product tryptophan efficiently (Fig.1.2).

Here two types of inducible promoter, Pλ promoter and lacUV5 promoter ,are used, which can be induced by heat (>42℃) and IPTG respectively. When lacUV5 promoter activated by IPTG, the cells will enter the “proliferation” state; while Pλ promoter is activated by heat, they will turn into the “production” state for expression of aroG and trpBA.

In order to test whether each part are functional individually, our working circuit is divided into several devives.

In order to realize the family application of our project and the automatic control of production conditions, we have designed a cultivation device. It contains controlling, detecting and cultivating modules (Fig.1.3). The equipment can monitor the cell growth and tryptophan production while conducting fermentation culture, and control the induction culture conditions through singlechip at different stages with this signal, so as to activate the expression of specific genes in the gene circuit, controlling the cells into “proliferation/production” state.

Considering it is difficult to realize real-time detection of tryptophan concentration by chemical method in hardware, we also have designed a detecting circuit which can sense the concentration of tryptophan and report green fluorescence of different light intensities (inversely proportional to the concentration of tryptophan) (Fig.1.4). By introducing this circuit into the engineered bacteria, the concentration of tryptophan in culture medium can be converted into light intensity that can be detected by hardware.

Through the interaction of hardware circuit and gene circuit, we can achieve both the automatic control and the high production, giving a full play to the advantages and potential of Synthetic Biology.