Project Outlines
This year Manchester-Graz team is trying to develop a whole new system for L-DOPA/dopamine treatment by regulating the pathway in an innovative way.
Fig 1 Administration of dopamine via gut bacteria
Low dopamine levels are linked to Parkinson’s disease, Alzheimer’s disease and several other health problems. The current treatment involves L-DOPA which crosses the blood-brain barrier, and is then enzymatically converted into dopamine. Studies show that Escherichia coli, Bacillus subtilis, Bacillus cereus, Bacillus mycoides, Proteus vulgaris, Serratia marcescens, and Staphylococcus aureus synthesize dopamine and that varying levels of dopamine in the gut can have neurological effects. Having bacteria in the gut producing dopamine would abolish the need for traditional medication, and only a single dose of culture in probiotics containing strains of self-regulating DOPA-producing bacteria will be needed. This is especially convenient for elderly people suffering from these diseases as there will be no such problem as an overdose or missing a medicine intake. This bacteria-based controlled drug delivery system incorporated could potentially improve the lifestyle of patients (Fig 1).
Fig 2 Novel dopamine expressing pathways
We plan to develop an innovative multidimensional cell density dependent auto-regulation system that could be used to control multistep enzyme pathways. L-DOPA is a good example as it is nowadays produced in bacterial cultures to cover the demand needed all over the world. The aim is to make the first steps in the development of a novel technology of drug delivery by developing self-regulating drug producing bacterial strains that, in the future, could be incorporated into patients’ intestinal and gut microflora to secrete medicines directly inside patients’ bodies in the response to any physiological changes, in particular low levels of dopamine.
Team Manchester is focusing on dopamine and L-DOPA biosynthesis. Two biochemical pathways will be worked on (Fig 2).
The team will identify the nature occurring enzymes for the biosynthetic pathways of L-DOPA and dopamine. Once biochemical pathways are proved to be credible, the genes of interest will be cloned into E.coli and correct synthesis will be further analyzed.
Graz-located team will use two quorum sensing based mechanisms for an autoregulated and time shifted consecutive induction of protein expression. This is first demonstrated and visualized on the example of fluorescent protein synthesis (Fig3). The fluorescent protein could then be exchanged with genes for the L- DOPA production in a way that at low cell density levels phenylalanine synthesis will get channeled.
Fig 3 Fluorescent protein synthesis dependent on different levels of cell density
After a certain biomass is reached actual L-DOPA production will be induced. As a future outlook this system could be further utilized to activate suicide genes in E.coli to avoid possible overgrowth of the native intestinal flora. We want to show proof of concept not only in E. coli strains, typically used in academic research, but also in strains that naturally occur in the guts. Therefore we plan to implement our system in E.coli Nissle 1917. Even though we cannot regulate the proliferation of our engineered strain yet, it allows us to provide an outlook of a possible application as a self-regulating drug mill in the GI- tract, that might be used in medicine in a couple of years.
Other potential applications for this project include in situ cell density measurement directly inside the bioreactor. Furthermore, this regulatory system allows human independent induction of protein expression by exchanging the fluorescent protein genes with the corresponding gene of interest. From an economical point of view this system circumvents the issue of expensive inducers like IPTG.
A main part of our project is also thinking about ethical problems, sociological aspects and human practice. These topics will be discussed in detail with high school students after talking about Synthetic Biology. In surveys, questions about public opinion concerning Synthetic Biology and genetic engineering will be asked. Survey data constructed by these discussions will be compared between Austria and the UK. Our project will make the first step to provide patients with drug-independent, novel- delivery and alternative-pathway technology to tackle neurological diseases. Additionally it is designed to allow a vast public outreach to show people advantages and benefits of Synthetic Biology.