Team:SCUT-FSE-CHINA/Description

Project Description

In the process of industrial fermentation, the problem of microbial contamination is always a tough challenge to face, which occasionally occurs due to the negligence of staff and equipment failure, resulting in very serious industrial accidents. Bacteria and phage infection control are the keys to the success of fermentation. The project aims to construct an E. coli dominant engineering bacterium by means of synthetic biology, which can prevent the contamination of bacteria and phage infection during the fermentation process, so that the fermentation can be carried out smoothly according to the production plan.

Part I: To solve the problem of bacteria contamination in the fermentation process

In order to resolve the problem of microbial contamination in fermentation process, we have constructed a new type of engineering E.coli possessing formamidase gene as well as phosphite dehydrogenase gene. The new engineering E.coli can utilize nitrogen and phosphorus source by hydrolyzing formamide to ammonia and oxidizing phosphite to phosphate for its own utilization and growth. Consequently, the basal MOPS medium in the presence of formamide and phosphite, but lacking of the basic nutritional components of \(NH^{4+}\) and \(HPO_4^{2-}\), makes the engineering E.coli grow and multiply while other microbes will be "starved" to death due to have no ability to assimilate formamide and phosphite, resulting in a solution to the problem of microbial contamination in E.coli fermentation process.

introduction

Part II: To solve the problem of phage infection in the fermentation process

CRISPR is a system for bacteria protecting itself against viruses. When the bacteria were resistant to exogenous DNA such as phage, CRISPR will be transcribed into a long RNA precursor (Pre RISB RNA, pre-crRNA) under the control of the leader. In addition, trans-activating crRNA (tracrRNA), which is complementary to the repeats of pre-crRNA, is also transcribed, stimulating the Cas9 and double-stranded RNA-specific RNase III nuclease to process pre-crRNA. After processing, crRNA, tracrRNA and Cas9 form a complex to identify and bind to the complementary sequence of the crRNA, and then cleave the DNA double strand to form the R-loop, making the crRNA hybridize to the complementary strand, while the other strand stays the free single stranded state. Then the DNA strand of the crRNA was cut by the active site in Cas9, and finally the DNA double strand breaks (DSB) were introduced. The cleavage site of CRISPR / Cas9 is located at the NGG site in the PAM region adjacent to the downstream of the complementary sequence of the crRNA, and this sequence repeat once each random DNA sequence of 128bp. On the basis of the bacterial CRISPR immune system, we hope that large fermenters can be vaccinated to prevent fermentation failures caused by phage infection in the environment. Therefore, our project envisages the rapid use of the CRISPR system to promote adaptive immunization of bacteria by selecting the types of phage that are common in the fermentation of E. coli and assembly their spacers together in a modularized manner to respond to the corresponding functional virus.

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