Self-regenerating, suspended-phase whole-cell biosensor system employing micro-chemostat and cell engineering technologies

We aim to make a mini device that can detect chemical weapons by containing living cells that have been designed to turn fluorescent green in the presence of harmful chemical warfare agents. The device will both keep the cells alive indefinitely and also be able to detect the level of fluorescent green colour they produce in order to measure the concentration of the chemical weapons.
Chemical weapons remain a danger in mainland Europe as a legacy of World War II and today the governments of several countries, including Belgium, France, Germany, and the United Kingdom remain actively engaged in locating and destroying old weapons and unexploded ordnance. Outside Europe many countries, ranging from developed (USA) to developing (Angola) typically retain chemical weapons capabilities measured by tonnage. As such there is an increasing need to detect the presence of chemical warfare agents in a manner that is sensitive, robust and reliable enough to safeguard as many lives as possible.
Currently most devices used by the military to check for the presence of chemical weapons work by using non-living material such as small chemicals, DNA or antibodies to detect chemical weapons in similar way to how pregnancy tests work.
Drawbacks to this 'molecule-based' approach are limited sensitivity, shelf-life and limited flexibility due to the need to often combine recognition of a particular chemical and detection within the same molecule. Living cells can detect compounds with extremely high sensitivity, the most famous example being sniffer dogs, but even their highly-sensitive sense of smell is eclipsed by that of bears who can reportedly detect food 18 miles distant.
We seek to directly harness the detection abilities of cells by actually having living cells resident within our proposed detection device. We will, for the first time, integrate three different technologies to build a 'living biosensor' device:

- Bioreactors allow cells to be grown and stimulated in a finely defined manner by providing a closed, controlled environment. This is used at large scales to grow cells to high density when making biological medicines such as hormones or antibodies. We have miniaturised bio-reactors down to a 'micro-' scale where controlled-environments cell growth chambers can be added to desktop or laptop-sized devices.

- The patterns of genes within cells can now be re-designed to make networks to give cells abilities chosen by the designer. We propose designing such a gene network that allows living cells to function as a component of a desktop or laptop-sized device. The 'synthetic' gene network will enable the cells to respond to the presence of chemical warfare agents by producing a fluorescent green protein or, in future, any other response that best suits the overall design of the detection device.

- The miniaturised, 'micro-scale' bioreactor will also require microscale plumbing and electronics to enable it to keep the cells alive and reliably detect levels of the fluorescent green protein. This requires cutting-edge micro-scale fabrication technology to make components at this scale for the first time.

Combining these three approaches in a unified goal of improved worldwide chemical weapons detection is a new approach and the technical insights that come from our efforts is likely to underpin many other new applications of these technologies in areas such as medical diagnosis and detection of contamination in food, medicines or clothes manufacture.

We propose a novel self-regenerating, suspended-phase whole-cell biosensor system for the sensitive detection of Chemical Biological Warfare (CBW) agents. The system is based on multiplexed continuous culture micro bioreactors (micro-chemostats) with cells in suspension engineered de novo to detect bio/chemical agents with high sensitivity. To achieve 'well-mixed' microfluidic reactors in a compact format we will use a minimal-moving-parts technique employing compact electromagnets close to the chamber floor actuating a small stainless-steel ball within the chamber that is cycled between points above each electromagnet. Microcontroller sequencing will drive the ball through the problematic perimeter region to achieve sufficient mixing and eliminate stagnant zones. Grouping electromagnets around the perimeter will render the central region accessible for optical monitoring. Cell-based detection systems comprise a synthetic gene networks in which levels of target agent directly drive expression of a reporter, and a 'threshold' comparator network in which any target encounter events trigger increasing expression of a reporter by a positive feedback loop. This synthetic biosensor system enables us to investigate the dynamics and robustness of synthetic gene network performance as a functional component of a microfluidic detection device. Accordingly, this proposal will harness current and future advances in Synthetic Biology and the translation of these advances into a wide range of novel detection assays. The biosensor detection system will be multiplexed using a fabricated duplex demonstrator with compact opto-electronics. This will allows us to progressively address the key challenges of microfluidic and optical read-out parallelisation which are paired with the requirements for robustness and compactness.

UK Public sector: This proposal offers the prospect of providing significant operational and economic benefit to the UK Armed Forces in the medium to long term by increasing the performance and reducing the whole-life costs of CBW detection devices and reducing the risk of CBW casualties in the field. MoD spend is under significant downward pressure due to the global economic downturn, with major implications for equipment spend. A sensitive, new generation, detection platform with a an extended active lifetime compared to existing cell based technologies would represent significant potential cost benefit compared to repeat purchases of single use devices. UK civil defence provision would also be enhanced by more compact, sensitive, new-generation detectors. Design options would allow increased sensitivity to be traded within system budgets to reduce size, power consumption, reagent consumption and noise level of collectors: all facilitating deployment in public arenas.

UK Bioscience: This project enables UK SMEs to reduce manufacturing costs of makiong biosensor devices and strengthen their global competitiveness and help lead the UK economy back to growth. A strong biologics manufacturing sector is currently driving recovery from the global economic downturn in EU member states such as Ireland. A similar turnaround is needed in the UK. By demonstrating sophisticated microfluidic and biological control of P. pastoris, this project will further leverage the impact of ongoing DN collaborations with UK SMEs, Cogent Ltd, BJS Technologies and Cyplasin Bioscience UK Ltd into P. pastoris host cell capabilities and ultrafast DNA synthesis techniques.

Third Sector: This work will contribute to increasing global security and so help fulfil WHO and UNICEF remits to promote international development and protection of vulnerable populations. Enhancing the capabilities of the UK military will feed into the ability of organisation such as the UN to safeguard combatants and local vulnerable populations. This will expand the number of regions with sufficient security to admit humanitarian activity delivered by local administrations and international NGOs.

General Public: This proposal will improve public and media perception of synthetic biology by providing a high-profile example of a practical application. The longer the absence of practical applications for synthetic biology, the more newsworthy it will be to label it as either over-hyped or dangerous (or both, as public opinions can often be contradictory). The practical achievements and benefits of this work will be communicated in a synthetic biology context through a range of public engagement activities such as UCL's regular participation in the international iGEM competition