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Revision as of 19:41, 17 December 2020


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Description


Presentation video

NANOFLEX: One Cellular Biosensor System for Many Applications

The Uppsala 2020 Team likes to think big. After rounds of ideation and brainstorming, we decided to dedicate our iGEM year to develop one sensor that could be adapted to many applications. Our project is built in stages, starting out simple, improving it afterward. This creates a steady ground for the development of NANOFLEX. What we present currently is a modular biosensor applicable for detecting a small molecule of choice and the steps to reach further applications like protein detection. You can read more about it below and on our poster.

What are Cellular Biosensors?

Cellular biosensors utilize the machinery of living cells as a detection system through genetic modification (1). In other words, we can manipulate cells, such as bacteria, on a genetic level to detect "things" (analytes) for us. The modified cells can identify specific analytes and generate measurable signals. The analyte is detected by a recognition element, like a receptor protein of the cell. Upon detection, a signal is transduced activating molecular paths to produce a visible or electronic output signal. (1) Analytes can be chemicals, pollutants, pathogenic substances or any other molecule that can be detected by the recognition element. Due to the vast selection of receptors and other specific proteins, cellular biosensors are an ideal tool for a broad range of applications, such as drug evaluation, environmental pollutant screening and diagnostics. (1)

Flexible Nanobodies: The Principle of the System

What sets NANOFLEX apart from many cellular biosensors is a modular detection domain, based on interchangeable nanobodies. Nanobodies are smaller than antibodies, but they still hold similar levels of affinity to their targets (2). They are also easy to clone, express and manipulate. Not to mention that they avoid traditional antibody manufacturing, stepping away from animal use in science. In our system, nanobodies are fused to proteins that induce a response signal in a cell upon nanobody-target interaction. The use of this system is not new, previous iGEM teams i.e. team Washington 2019, team NTHU Formosa 2018 as well as several papers have brought up the opportunities laying within this design i.e. Chang et.al 2018, A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria (3).

Development of NANOFLEX

Focus: a functional and iGEM standardized detection domain. Theory is great but we had to put the system to test. We started with an easily accessible target, caffeine. Replicating the work of Chang et.al (3) we used their caffeine-detecting camelid domain, but together with a quick reporter alternative, the monomeric Red Fluorescent Protein (mRFP), to attempt building a testable system. Which we succeeded with - you can read more about our assays and modelling in proof of concept, and find the list of parts of this system here. In future/implementation you can read more on developing new nanobodies for NANOFLEX.

Improving the Design

To further optimize the design of NANOFLEX we researched for reporter, noise reduction and signal amplification systems. On our design page you can read more about our reasoning behind choosing β-galactosidase as the reporter enzyme, T7 RNA polymerase system for controlled signal transduction and Qβ viral replicase as the signal amplification agent. As the design started to include more components and the genetic burden of the components started to weigh in, we also developed a new set of low copy number plasmids to fit our BioBricks. Read more about them here.

Modular Assembly for a Modular Design

The preferred assembly system throughout our project was iGEM Type IIS standard. Its modularity bedded well for an easily modifiable system and the switching between components. As one of the first teams working with this system, we faced some issues regarding the lack of standardized approaches to Type IIS, thus we developed a guidebook we hope will be of great help for future teams.

Exploring Protein Detection

To further allow the system to be applicable on protein targets we researched how to expose the detection domain on the outer membrane of a model organism. Thus, we explored the alternatives on either removing the outer membrane of Escherichia coli, as well as transferring NANOFLEX to a gram positive model organism Bacillus subtilis. Our research on Bacillus subtilis and bacterial cell walls is also compiled in our Contribution page for the benefit of future teams working with this model organism.

Application: Beyond Wet Lab

When envisioning NANOFLEX as a commercial product we followed World Health Organizations ASSURED criteria. A-Affordable, S-Sensitive, S-Specific, U-User friendly, R-Rapid and Robust, D-Deliverable to the point of care (4). These criteria have been closely integrated in our molecular design but every application sets different needs for example for deliverability and user friendliness as end users may vary.

Case study

To explore the plausible implementation of NANOFLEX we worked on a case study of the system. The disease we chose was Tuberculosis, because despite the fact of Mycobacterium tuberculosis being an ancient bacteria menacing humanity for a long time, we still struggle with timely detection of it. Our literature research showed HSP16.3 being a suitable biomarker to target. You can read more about nanobody development for this biomarker in dry-lab and more on our exploration on Tuberculosis in Integrated human practices.

Making NANOFLEX More Accessible

To be able to deliver NANOFLEX to the point of care in such applications we had to reason on how deliverable our kit would be. Hereby we tested lyophilization of our bacteria, read more about it here. To make the sensor more available we developed a prototype of a commercial product, read more about the hardware here.

Making Knowledge More Accessible

Our motto while developing NANOFLEX was always “Making knowledge more accessible and not reliant on expensive detection instruments or expertise”. We held behind it. Thereby in parallel with everything above we took the chance on spreading the science in general. Read more about our public engagements, education efforts, and collaborations.

References

  1. Asphahani, F., and Zhang, M. (2007) Cellular Impedance Biosensors for Drug Screening and Toxin Detection. Analyst. 132, 835–841
  2. Huang, L., Muyldermans, S., and Saerens, D. (2010). Nanobodies®: proficient tools in diagnostics. Expert review of molecular diagnostics. 10(6), 777-785
  3. Chang, H.-J., Mayonove, P., Zavala, A., De Visch, A., Minard, P., Cohen-Gonsaud, M., and Bonnet, J. (2018) A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria. ACS Synth. Biol. 7, 166–175
  4. Kosack, C. S., Page, A.-L., and Klatser, P. R. (2017) A guide to aid the selection of diagnostic tests. Bull. World Health Organ. 95, 639–645