Overview

Trace substance detection remains challenging, especially regarding food safety and medical care in daily practice. While it is possible to examine substances with great accuracy in labs carefully, it’s usually too costly or inconvenient to conduct it in everyday life.

And NOX is here to settle it.

We chose two examples to demonstrate our ideas.

Bisphenol A (BPA) is generally presented as an additive in plastics. With a structural basis resembling estrogen, it possesses the possible influence of disrupting estrogen receptors, leading to damage to the reproductive and immune systems.

Liver dysfunction is a global healthcare burden, affecting the life quality of millions of people. In vitro sampling diagnostics is only available for hospitals, while its chronic nature requires daily monitoring. Bile salts are critical indicators of liver disease, and their detection is vital.

This year, we designed and proposed NOX (Neo-quOrum sensing-based Xpression biosensor platform) to provide a highly compatible and robust platform with impressive performance, taking advantage of synthetic biology to construct an artificially organized signaling pathway.

Chimeric receptors are assembled for optimal compatibility, and the orthogonal quorum sensing module is responsible for luminescence, optimized by modeling. Our hardware collects and processes the signal, which is readily accessible for further analysis.

We also provide a portable and scalable device with a meager cost, aiming to offer civil detection covering multiple uses.

Why whole cell biosensor

Bacteria possess inherent sensory capabilities essential for their survival and reproductive processes, as they must detect and react to a diverse range of chemical and physical stimuli. Whole-cell biosensors (WCBs) are a type of bacteria that have been genetically modified, which are even better than naturally skilled bacterial sensors, possessing an impressive performance.

With the assistance of synthetic biology, whole-cell biosensors can respond to extremely low concentrations of target molecules, covering a wide range of similar structural chemicals with high specificity. Moreover, whole-cell biosensors possess the inherent capacity to offer compact, easily transportable diagnostic apparatuses that can detect many targets simultaneously and perform intricate computational tasks.

Two strategies and the Cascade

In order to address the problem comprehensively, we propose two strategies for biomarker or trace chemical detection, and we take advantage of orthogonal quorum sensing to minimize the influence on regular metabolism and other pathways and seek to maximize the signaling effect.

The first strategy is creating chimeric receptors based on single-domain ligand binding domain(LBD), which could be directly extracted from transcription factors or nanobodies. We use bile salt, a general and reliable biomarker for liver dysfunction, as an example.

The second strategy is signaling through degradation. When finding relevant transcription factors is tricky, we could first degrade the target molecule and amplify it through exogenous receptors.

Demonstration of the Two Strategie

Orthogonal Quorum Sensing is based on an imported system of LuxI/LuxR from Vibrio fischeri. Taking advantage of the mutant of LuxR, which is specific to VAI, we eliminate the possible effect on the chassis bacteria. We use Nanoluc as our output, which performs well upon signaling(as shown in the Results). It will also be more accessible for our hardware to analyze the output.

Altogether, we build a cascade based on our first messenger - the molecule of interest, and the second messenger-VAI.

The Constructed Cascade

Drylab

In order to support the NOX project, this year Drylab focused on designing from three phases of the iGEM experimental process:

• For project verification, we verified the theoretical feasibility of NOX through modeling and simulation, providing theoretical expectations and validation for experiments. Our modeling predicts and validates the results, with analysis of experimental data. Our modeling results also provided guidance for the reaction chamber design by the hardware team. Based on experimental feedback, we performed fitting to optimize and analyze the model. (See Model for more details)

  Structure of Modeling

• For project construction, we developed the NOX Kit biological detection kit (See Hardware for more details), aimed at enabling NOX to more easily transition from the biology lab to broader usage environments, providing low-cost reliable experimental testing environments for users, experimenters and developers.

  A sketch of NOX Kit Structure: The detector array in the figure consists of multiple NOX Kits connected via an I2C bus and communicating through a host and PC. The sensing component of each NOX Kit is composed of a photosensitive resistor and thermistor, which not only enables high-throughput detection through cluster operation, but also allows independent operation for convenient sensing. Furthermore, the NOX Kit is a low-cost detection device, with each unit costing less than 1 USD.

• For project pre-research, we developed a natural language query program for the Biobricks database called Ask NOX (See Software for more details), aimed at reducing the pre-research time finding suitable Biobricks from the massive Biobricks database.

  Demo of Ask NOX: Simply input description for Biobricks that you nead, and Ask NOX will return the most relavent Biobrick according to your natural language inputs.

Safety

We considered three different modules for biosafety and conducted careful procedures for hardware safety. We are introducing one biosafety method here, while other methods utilize unnatural amino acids and temperature control in the chamber. For more detailed safety information, please click here.

In the IPTG experimental setup, the antidote is synthesized within the device and subsequently interacts with the Doc toxin. Upon release from the device, the toxin exerts its bactericidal effects on the external bacterial population.

Schematic Diagram of the Kill Switch

User Manual

Lastly, we have designed elaborate procedures to help our users better handle NOX to achieve their personalized detection goals suitable for different situations.

The Overall Guide of Using NOX Kit

Packaging

1. Sealed vials containing a fixed amount of lyophilized bacterial powder (re-sealable)

2. IPTG water solution of appropriate concentration

Dectection

To perform a detection test, follow these steps:

1. Obtain the detection sample in liquid form. If the sample is not in liquid form, dissolve it in pure water to make a uniform solution.

2. Open the product packaging and the sealed tube containing the bacterial powder. Add IPTG water solution to the tube.

3. Shake the tube gently to make the solution uniform and revive the engineered bacteria.

4. Cover the tube and place it in the detection device.

5. To identify the results, check the fluorescence intensity displayed on the device. The result can be told from the intensity change compared with the negative control group.

6. To ensure biological safety, use ultraviolet radiation or medical alcohol to wipe the detection equipment after each use. Soak the tube containing the waste bacterial liquid in chlorine-containing disinfectant for at least 10 minutes to sterilize it. For centralized treatment, use high-pressure steam sterilization. Dispose of the sample tube according to medical waste disposal guidelines.