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.

Fig.1.1

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.

Fig.1.2

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.

Fig.1.3

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.

Fig.1.4

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.

Experiment design

We designed and construct two circuits to verify the function of aroG and toggle-switch circuit respectively as first step.

aroG

To release the feedback inhibition of phenylalanine on aroG and improve the catalytic efficiency, we mutated serine at site 211 of aroG into phenylalanine to obtain its mutant, aroG-S211F (Part:BBa_3832000).

We have constructed a circuit (Part:BBa_K3832008) for verifying function of aroG-S211F (Fig.2.1). lacUV5 promoter is used to make the expression of aroG-S211F inducible, which facilitates our measurement.

Fig.1.5

Toggle-switch

GFP and RFP are used to help us detect and check whether our design of toggle-switch circuit will work properly (Fig.2.4). Each promoter downstream follows another promoter's repressor gene and a fluorescent protein in different color. By detecting the relative fluorescence intensity under different inducing conditions, we can get information about the intensity of each promoter in different states and whether they can function as we designed.

Fig.1.6

Result

Verification of aroG-S211F

We have measured the yield of tryptophan and proliferation rate in the chassis, E.coli DH5alpha, containing this circuit, and by comparing with corresponding control groups (without aroG-S211F or not induced by IPTG), we have successfully verified its function of promoting tryptophan production and inhibition effect on cell proliferation (Fig.2.2 & Fig.2.3).

Fig.2.1

More details for the experiment design and result of aroG:
Improvement: aroG-S211F

Fig.2.2

Verification of toggle-switch circuit

Our experiment result have confirmed the feasibility of our design. As shown in Fig.2.5, we successfully achieved bistable protein expression under different induction conditions. The relative expression levels of GFP and RFP were significantly reversed with the change of inducting conditions, indicating that the circuit design can be used to switch cell states between “proliferation” and “production”.

Fig.2.3

More details for the experiment design and result of toggle-switch circuit:
Engineering Success: toggle-switch circui

Conclusion

By detecting the functions of aroG and toggle-Switch circuits respectively, we verified the feasibility of our general design. We confirmed that the expression of exogenous aroG-S211F can significantly promote the production of tryptophan and observed its inhibitory effect on cell proliferation. Meanwhile, for the control circuit, two groups of promoter-repressor systems and RBS with corresponding strength we used can achieve bistable state under different induction conditions.

Although we did not verify the performance of aroG-S211F, trpBA and pykA genes in toggle switch circuit, based on the data obtained in the experiment, we also confirmed its feasibility through modeling (View results on our Modelling page). Furthermore, we predict the best time to change the induction condition when the tryptophan production reaches the maximum, so as to provide suitable parameters for automatic control of hardware.