Parts for a reliable and field ready biosensing platform

Judging Form

Abstract

Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field. We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

Introductory Video:

Background

Electronic circuits excel at logic and speed of computation, but aren't particularly flexible when it comes to sensing the wide-range of small molecules present in the world. The use of biosensors as an approach to determining concentrations of analytes in samples for a huge variety of applications (medical, doping, ground water contamination - See Human Practices) has great potential to be revolutionary technology.

Currently, however, whilst plenty of biosensors exist and have been characterised to a greater or lesser degree, they are in no way unified or standardised. Not only that, they are almost exclusively either non-quantitative or only usable in the lab, read using expensive and delicate laboratory equipment. They are also limited, as is often the case in synthetic biology, by the stochasticity of life, often giving unreliable or poorly reproducible results. Furthermore, at present relatively little thought has gone into the storage and distribution on biosensors for use in the field where shelf life, cost of transportation, storage conditions and biocontainment all become important factors.

We aim to develop parts towards a new standard for biosensors that addresses all these issues. The standard is to be back-compatible, so not only will new biosensors developed with the standard have these properties, but so will pre-existing sensors after minimal adaptation.

In order to develop our final system we went through a comprehensive Design Process.

The final result of our standard design is outlined below and consists of four parts:

Overview of Systems

Ribosense

We are investigating new and novel biosensors. We have decided to use riboswitches, as they are under-represented in the registry and have the potential to be widely used in the future. We have decided to work using two riboswitches, one for fluoride, and one for magnesium, as they have opposite mechanisms and so are representative of potential future ribosensors.

Ratiometrica and use of bacterial luciferase

Biosensors may give unreliable outputs. This is due to differences in the number and state of the cells from test to test. By including an internal control signal, to which another inducible signal may be normalised, the reliability and reproducibility of a sensor may be significantly improved. We are currently working on two such two-signal systems.

Firstly, a construct that uses an inducible eCFP and a constitutively expressed eYFP. All components, save the vector, are existing biobricks. This will serve as a proof of concept and a way of testing old and new sensors. However, this will require a platereader to use.

The second system is based on luciferase and an mOrange/luciferase fusion. Luciferase light emission is visible to the naked eye, and can therefore be sensed and quantified using inexpensive, off-the-shelf electronic components, giving it an advantage over fluorescent proteins in this context.

Development of a cheap and easy sensing kit - Biologger

We have developed a cheap kit for digital quantification of the lux-based construct. Developing cheap, functional bio-electronic equipment is crucial for the development of synthetic biology into an industrialised field. All components are inexpensive and readily available. It is self-contained, yet modular, allowing for customisation. It is based on the Arduino microcontroller, and is compatible for use with a PC or an android smartphone, for ease of use in the field.

Sporage and Distribution

Bacillus subitilis forms long lasting dormant spores, which can be kept at room temperature in a sealed vessel. Compared to E.coli, which must be kept in a freezer or freeze-dried for transport, the practical benefits of using subtilis are self-evident. We have isolated genes in subtilis that can be overexpressed to improve spore germination rate.