Team:UTK-Knoxville/Project

Motivation

   A major problem in modern biology is a lack of well understood microbial sensors and one limitation in metabolic engineering is an inability to control dynamic cell metabolism. Our project provides a solution to both problems with synthetic biology via a synthetic sensing scaffold. There are no standardized platforms for sensor characterization today, and as synthetic biology grows, so does the need to develop standards for the creation of genetic construct. The completion of this project provides a high throughput method of developing a library of microbial sensors, a mechanism for controlling cell metabolism based on environmental conditions, and a standardized platform for the design and characterization of synthetic sensing circuits. This project aims to introduce a “plug-and-play” method for creating sensing circuits, and to provide a protocol for characterizing novel sensors made in this way.

What is a Chimera Protein?

   A chimera protein is created by fusing portions of multiple genes together via a technique known as overlapping PCR. In our case, we fuse two genes together in our chimera: a sensing domain and a histidine kinase. The sensing domain is the region responsible for binding to the target analyte, and the histidine kinase is activated by binding in the sensing domain and passes on a signal to a reporter. The histidine kinase EnvZ is used in our project and kept constant for each and every sensor that we build, making it highly modular.
    Once the scaffold has been built, the only entity needed to build a novel sensor is a sensing domain to pair with EnvZ in the chimera, which is then plugged into the platform that has already been established. The simplicity of the PCR needed to construct a new chimera makes it both quick and easy to build novel sensors using either known or unknown sensing domains, thus providing a high throughput method of developing a library of well characterized sensors. Introducing an optimal, standardized method to synthetic biology betters the entire research community and allows more efficient communication.

Platform

   Our platform is a two-plasmid system containing a sensor plasmid and reporter plasmid. The sensor plasmid contains a chimera gene and the transcriptional regulator OmpR. When the sensing domain paired in a chimera with EnvZ is activated, a signal is passed to EnvZ, which in turn activates OmpR; OmpR then upregulates the promoter OmpC via phosphorylation. In our reporter plasmid, a fluorescent protein is under the control of OmpC, meaning that only when the sensing domain is activated does the fluorescent protein being to be transcribed.
   This standardized method of building synthetic sensing circuits introduces is the first of its kind in synthetic biology, providing the research community with a uniform way of testing new sensing domains. Because the system can be manipulated efficiently and produce a predictable, consistent fluorescence response to any sensing domain built into a chimera, it helps to quickly address the lack of characterized sensing domains in microbiology today.

Reporter Plasmid

   Our reporter plasmid is highly versatile and can be manipulated to produce a wide range of signals in response so a chemical stimulus. Though we used red fluorescent protein, the response changed can be anything from a different fluorescent protein to beta-galactosidase by removing RFP and replacing it with another gene under the control of the transcriptional regulator OmpC. The circuit’s reaction to the analyte can be changed so that the response that fits the application at hand or the user’s desired measurement method.
   Because the response of the reporter can the transcription of any gene, the reporter does not necessarily have to produce a response such as color change. One option is to put a gene under the control of OmpC that has a direct effect on cellular metabolism, meaning that the system can be designed so that cellular behavior is synthetically changed based on environmental conditions. This has immense potential in the field of metabolic engineering, as it would be the first sensing circuit developed that can alter cellular metabolism in this way.

Proposed Sensors

NarX-Nitrate and Nitrite

   The sensing domain NarX responds to both nitrate and nitrite. Nitrates are found in fertilizer and explosives, and can be hazardous to humans. Nitrites are found in acid rain and are also used in food packaging. Because of this, this versatile sensor has applications in both environmental observation and human health.

Aer-Oxidizable Substrates

   Aer responds to oxidizable substrates within the cell, but does not react with substrates outside of the cell. Because of this specificity, this sensor can be used in metabolic monitoring.

TodS-Toluene

   Toluene is a benzene derivative found in paint thinners and sealants. It can also be used as a mechanism of boosting fuel efficiency. It can cause extreme neurological damage if inhaled or ingested, so monitoring toluene levels has applications in human health and safety monitoring.

RCom-NO2

   Nitrogen dioxide and is known to be a major contributor to pollution and environmental damage, as it is a byproduct of internal combustion engines. It is also highly combustible and can be toxic when inhaled. It is a common intermediate in the the production of nitric acid, is found in high concentrations in places of nuclear testing, and is given off by many heating units. This sensor can also be used in environmental monitoring and to protect human health.