Team:BIT-China/Design

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Design

Transcription management is a cell regulatory system used in the fermentation industry.

We learned that E. coli has endogenous MEP pathway, which can introduce three another exogenous genes to produce lycopene. And lycopene is a colored compound that allows us to easily measure production. Finally, we agree to build the "Achieved Transcription Management" in E. coli.

To design a transcription resource allocation system for cells, we need to focus on analyzing the relationship between the specific gene transcription levels and time. The growth process and production process for bacteria need to be artificially separated according to time in order to maximize production and minimize growth. Therefore, we need to establish a system that can recognize and feedback the population density of the target bacteria.

Quorum sensing systems are often used to identify population density.

AHL-mediated communication is the most widely studied and the best understood model for QS ^[1] . We chose QS system from Vibrio fischeri to develop our system. From kits we found the parts that build Lux systems: LuxI, LuxR and P_lux.

Previous teams have point-mutated the P_lux promoter to change the induction threshold. We focused on analyzing the effect of LuxR protein concentration on this system.

When the transcription of target gene is controlled by T7 promoter, T7 RNA polymerase is very active. Endogenous RNA polymerase almost can’t take part in transcription. The σ factor and core enzyme are part of the RNA polymerase. The σ factor itself does not turn on transcription, but specifically pulls RNA polymerase to a specific promoter binding site. The σ factor orthogonal expression system has no cytotoxicity yet more promoters, a wide range of regulation, and weak leakage.

Experiments have demonstrated the orthogonality of σ factors ^[2] .

In fact, the expression levels of different intensity can be obtained by point mutation promoters. But in our project, we want to precisely regulate the level of expression of each gene in the metabolic pathway.

In the CCiC meeting, a judge pointed out that the expression of T7 was unstable and the experimental dispersion was large.

In addition, we hope to automatically regulate the amount of each enzyme by introducing a negative feedback circuit. In the original conception, we wanted to select different sigma factors and corresponding promoter combinations to control the expression levels of different genes. But we found that sometimes this does not perfectly balance the metabolic flux. Negative feedback may do the trick.

Lycopene is the product of our project to verify that the system works. The lycopene precursor IPP and DMAPP were synthesized by E. coli endogenous MEP pathway. The lycopene functional genes crtE, crtB and crtI were introduced into E. coli and induced by the orthogonal sigma factor and T7 expression system. Calculation of enzyme activity ensured the balance of metabolic flux.

Building a dual plasmid system requires validation of plasmid compatibility. We selected pUC19 and pET28A as plasmid backbone, and we introduced these two plasmids into E. coli to verify their compatibility.

The significance of this project is to adjust the contradiction between growth and production. We want the cells to grow first and then synthesize the product. If the system presses too hard on the cell growth process, the time cost will rise, which is against the original purpose of the project. We set up a control group for comparison, considering cell growth rate and production. And a mathematical model has been established to analyze the bacterial growth.