Difference between revisions of "Team:Oxford/Design"

Introduction

It was important to us that our design was as uncomplicated and elegant as possible. To effectively diagnose Chagas disease, we needed a system that could be performed in a field setting. This eventually pushed us in the direction of a cell-free system, inspired partly by the work of Keith Pardee in diagnosing the Zika virus cell-free. Synthetic biology presents many possible paths, and we chose to investigate two of them: one DNA-based, one protein-based. The DNA-based system uses the TetR repressor, and the protein-based system uses OmpA and SpyTag/SpyCatcher to tether proteins to the outer membrane.

Background

There are many issues associated with developing a diagnostic for a neglected tropical disease. The method has to be (1) effective, (2) affordable, and (3) viable to perform and store within existing infrastructure. Additionally it needs to be accepted by the local community as a reliable and safe method for them to use. Currently, diagnostics for congenital Chagas disease do not sufficiently meet these requirements. Synthetic biology is a currently underutilised as an option for diagnostics. Many tests use enzyme linked immunoassays (ELISAs), which can be expensive and difficult to conduct in some environments. They also rely on having common molecular epitopes, which may not be present for some diseases. Synthetic biology allows us to detect in a different way, and also circumvent the use of antibodies in some cases. It can also lead to smaller, faster, and cheaper detection. We conducted a survey of the general public and found that a majority were in favour of the development of a diagnostic using synthetic biology.

Cruzipain as a biomarker

Most current tests for Chagas disease focus on detecting the whole parasite, T. cruzi, its DNA, or antibodies produced in response to its biomarkers. We looked at a traditionally difficult biomarker, cruzipain(ref), a specific cysteine protease. Most tests failed to use it because it has many isoforms and no stable epitope, however its substrate was always the same, and we decided this was ideal to use for our system. This is because it is secreted by the parasite, and as the parasite is present in relatively high concentrations during the acute phase of the disease, there are likely to be relatively high concentrations of cruzipain present. It is also highly specific to T. cruzi meaning that we are unlikely to get false positives from this aspect. By looking at the protease directly we also circumvent a traditional problem which is diagnosis of newborn babies, who do not have fully developed immune systems and can therefore not use immunological tests. This developed our focus on congenital diagnosis. We developed two systems to detect it - one is DNA-based, one is OMV-based.

Design-Build-Test cycle

We used the engineering cycle from Imperial 2011 to help with the design, build, and testing of our system. This cycle shows how interactions with modellers, consumers, and the public contribute to the development of a system, and integrate with the wet lab work. The most important part is it is cyclical in nature, and a design may go through many cycles before it is finished. This constant dialogue is key to creating a product that matches the needs of the consumer as well as being scientifically sound. It allows for iterative improvement of the device.

First DBT cycle

Originally we envisioned producing hirudin directly from our system. However, results from our modelling data (see here) suggested that this would not be fast enough to prevent blood clotting. Thus, we needed an amplification system to increase the speed of hirudin production, and we settled on doing this by releasing it from a sterically hindered system by cleavage by TEV protease. From there, we considered the options of how we could control the levels of TEV protease in the system.

DNA approach

To detect cruzipain, its proteolytic function should lead to a detectable output. This leads to two logical options - a positive output and a negative output. As the system is DNA-based, and we want to control the production of an mRNA that would then be translated, this suggests either using an activating or repressing transcription factor. Cleavage by the protease to allow an activator to work would be a positive output, and cleavage to disable a repressor would be a negative output. After research, we concluded that it would be more feasible to produce a negative output using proteolytic cleavage.

Therefore we decided to research repressor proteins which could potentially be inactivated by cleavage from cruzipain. After reading several options, we decided on the TetR repressor(ref) as it was shown to be a very strong repressor, and had an easily accessible linker region between its two domains which we could modify to have a cruzipain recognition sequence.

TetR acts as a dimer(ref), and each monomer has a DNA binding domain and a dimerisation domain(ref)., consequently the Tet promoter has two TetR binding sites, both needed for full repression of transcription(ref). We took the DNA sequence of TetR Class B from BBa_K106669 and located the linker region using PyMOL and PDB entry 3zqi. The linker region was long enough to modify into a protease recognition sequence.

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