Team:Virginia United/Project

Quorum Sensing Amplifiers and a Co-design Approach for Information Processing

In engineering, it is recognized that there are various ways to successfully design a mechanism. Often, different designs are tested and compared given a set of metrics in order to assess the strengths and weaknesses of each option. This process is called co-design. Since synthetic biology is truly a multi-disciplinary field, it is important that we incorporate techniques that have been proven successful in other well-established disciplines. We decided to apply co-design while testing and evaluating the effectiveness of a multiple-compound biosensor detection process.

In order to compare our designs, we are implementing logic on three different regulatory levels of the cell. One approach utilizes the operator sites of regulatory promoters, hybridizing two promoters’ operator sites into a single co-sensing promoter. In order for the hybrid promoter to initiate transcription, both target compounds that control the operator sites must be present. The hybrid promoters are attached to a single fluorescent protein, so detection of both target compounds can be measured via fluorescence.

Another method utilizes fluorescent protein complementation. When a target molecule is detected by a cell, it will transcribe a non-fluorescent half of a protein. When another target molecule is detected, the other half of the fluorescent protein is transcribed. Upon translation, the halves will bond together and fluoresce, reporting the presence of the two target compounds.

The final approach allows each of the sensory reporters to express a fluorescent protein in the presence of its target compound. If multiple target compounds are detected by a culture, fluorescence spectroscopy is used to separate out the wavelengths of each fluorescent protein, which then determines what compounds are present in the system. In all three designs we are amplifying the signal that each E. coli cell emits once it is exposed to the target compound with a quorum sensing system. Each cell releases a signal when exposed to the target compound, which is then recognized by neighboring cells. A fluorescent protein is attached to the promoter that recognizes the signal, establishing a more rapid, binary-like response time in the system.

We decided to evaluate our co-designs by developing a biosensor that detects the presence of mercury, copper, and arsenic in aquatic environments. In small doses each metal may not individually be toxic to fish, but a combination of the metals, even if individually each metal is at a negligible volume, may still be hazardous to both the fish in such environments and the people that later consume those fish.

As a final product, we hope that the system can be utilized not just for testing aquatic toxicity levels, but can serve as a basis for a synthetic machinery that has interchangeable inputs and outputs so it can be used for other applications such as biosecurity. Additionally, with an interchangeable response, it is possible to not only implement a biosensor within this system, but in the future, bioremediation machinery could be applied as an output as well.

Project Details

Abstract

Synthetic biology endeavors to create information processing systems modeled on digital electronics. The use of quorum sensing can help transform an inherently analog molecular signal into a binary response and simultaneously allow the tuning of input response thresholds and signal amplification. This project demonstrates these capabilities through experimentation and modeling. Another candidate for reapplying an electronic engineering technique is the codesign of hardware and software to implement a function. In synthetic biology, codesign might mean implementing a design spec in different expression control regimes and comparing their relative merits. Our work examines the codesign concept by constructing an AND gate in three different design domains. We explore the application of these ideas with an environmental sensor. A unique aspect of our project is the collaborative nature involving five institutions at three locations, which fostered a codesign-like approach using two distinct assembly techniques.

Overview

As the field of biomedical engineering grows, so too does the complexity of genetically engineered systems. Fabricated metabolic pathways are becoming increasingly intricate and expansive: how is one to know if the approach used to synthesize a pathway is necessarily the best approach? Our group, composed of five institutions, strives to show that co-design, a common engineering practice used to evaluate relative efficiencies, can be implemented to select the best design for a system. Using a biosensor as an application of carrying out co-design, we constructed three systems utilizing different mechanisms that can detect inputs and give an output correlating to which input(s) it senses. To maintain consistency, our systems are designed to use quorum sensing to amplify signal transduction upon input detection. This essentially acts as an on/off switch, allowing the system to be engineered to detect a certain input threshold and establishing a binary response.

Quorum Sensing

Normally, quorum sensing is a mechanism employed by cells in which signaling proteins are released and passed on to other cells in order to communicate certain information or to encourage other cells in the vicinity to act out certain functions. In our experiment, we wanted to utilize quorum sensing so that when the E. coli cells sensed that there was a input in the environment, it would not only produce some type of fluorescent response, but also initiate the quorum sensing mechanism so that the signal would be amplified and passed on to other cells that would then react and fluoresce as well. In that manner, instead of receiving a gradual response to the stimuli, we would actually receive a quicker, more binary response that would be easier to detect and measure. Thus in our experiment, we wanted to employ quorum sensing as an amplification mechanism that would provide us with binary output, making the system easier to manipulate into a quantitative response system.

Fluorescent Complementation

Magliery et. al (2004) developed a method for detecting protein binding partners by fusing non-fluorescent fragments of GFP to the two peptides of interest. If the attached peptides sufficiently attract one another, the GFP fragments will be irreversibly reassemble to form a fluorescing GFP protein. We build on their work by demonstrating how GFP fragment reassembly ("fluorescent complementation") can be used to create AND logic.

Magliery et al. Detecting Protein- Protein Interactions with a Green Fluorescent Protein Fragment Reassembly Trap: Scope and Mechanism. J. Am. Chem. Soc (2005) vol. 127 (1) pp. 146-157

Hybrid Promoters

The advancement of synthetic biology has opened the doors to almost limitless transcriptional control, given the particular biological species cooperates. The aim of the hybrid promoter circuit is to give one fluorescent output based on any two inputs. To do this, logic AND gates are designed using promoters and protein operator sites.

Using literature, current biobricks, and promoters J23106 and J23110 as base promoters, two sets of promoters were designed to respond to tetracycline and lactose, tetracycline and arabinose, and lactose and arabinose. The promoters are predicted to have similar transcription rates as their base promoters. According to literature, a promoter with an activation site that is not activated is not entirely inactive. RNA polymerase can still bind, only the interaction is weaker. Promoters J23106 and J23110 were chose because they are of relatively moderate strength. It is predicted that until arabinose is present in the system, the relative strength of these promoters will be fairly weak. When coupled with weak ribosomal binding sites, the rate at which proteins are translated should be negligible, preventing false positives. Also, following each fluorescent protein, is the respective quorum sensing protein to keep the system active.