Problem

Parkinson's Disease (PD) is the fastest-growing neurological disorder in the world. It's the second-most common neurodegenerative disease after Alzheimer's disease, affecting more than 10 million people worldwide (1). Symptoms are mainly a result of the degeneration of dopaminergic (dopamine-producing) neurons in the substantia nigra region of the brain due to the aggregation of misfolded α-Synuclein protein. Patients suffer from motor (tremor, rigidity, slowness of movements and more) and non-motor symptoms (depression, anxiety, gastrointestinal disturbances, loss of smell and more). Before motor symptoms, which are the most well-known, are evident, more than 50% of the brain's dopaminergic neurons are already lost (2).This is irreversible as of now and leads to a diminished response to treatment and thus a worse prognosis.

A cure does not yet exist, but earlier intervention has been shown to improve the course of the disease in several studies (3). Patients have the opportunity to work on lifestyle choices that have a positive impact on PD symptomatology, like diet and exercise. It would also have a multiplicative effect on the efficacy of any future treatment, possibly stopping the disease in its tracks. But how could we accomplish this? A relatively recent development may hold a clue, and also an inspiration.

Inspiration

In 2015, a retired nurse from Scotland, Joy Milne, made headlines due to her uncanny ability to detect Parkinson’s Disease by smell.

Due to her hereditary hyperosmia, she was able to notice a difference in her husband's odor 12 years before his official diagnosis, when he was only 33 years old. Her abilities were validated in the following years, and in 2019, several compounds largely responsible for the “PD smell” were identified. Out of these, four Volatile Organic Compounds (VOC) were found to differ in concentration in the skin sebum between healthy people and PD patients. These were eicosane, hippuric acid and octadecanal, whose amounts increased, and perillic aldehyde (perillaldehyde), whose amounts decreased in PD patients (5).

Thus, a new biomarker was found, and a target for us to detect. Up until now, this procedure was done with large, expensive and inaccessible Gas chromatography-Mass spectrometry (GC-MS) machines, and a cheaper, portable and easier-to-distribute solution was largely needed. And again, how could we do this?

Proposed Solution

Our proposed method for this detection is to use the M13 bacteriophage as a colorimetric biosensor. So, let us first discuss our organism of choice.

M13 is a filamentous bacteriophage of the genus inovirus, which infects E. coli that carry the F-episome. Unlike the vast majority of bacterial viruses (e.g. MS2, Qβ, λ and T7), active infection with M13 does not kill the host cell. The M13 phage particle consists of a single-stranded DNA (ssDNA) genome encased in approximately 2700 copies of a major coat protein, p8, and 5 copies each of 4 minor coat proteins: p3 and p6 on one end and p7 and p9 on the other. (6)

Its elongated form and pVIII proteins protruding from the side, when viewed from such an angle, are reminiscent of a centipede. Since its goal is to detect compounds associated with the “PD smell”, one could (but maybe not should) call it a SCENTiPD.

The technology we are using for our purposes has been described as the production of a "Phage Litmus" (8). It involves the deposition of engineered bacteriophages on a substrate, usually Au-coated, and the formation of specific hierarchical structures. These are generated and tuned by controlling the speed with which the substrate is pulled (vertically) from a bacteriophage solution of specific concentration. This pulling speed creates a dragging force between the air and the liquid surface, resulting in the said deposition and self-assembly of bacteriophages onto the substrate.

This technique utilizes the phage’s capsid proteins, which can obtain affinity for the target compounds through genetic engineering. More specifically, the target for modification is M13’s pVIII, or Major Coat Protein. This procedure has been tested on other volatile compounds, including TNT, or functions, like generation of piezoelectricity. (9)

After the VOC-pVIII binding has taken place, the phage bundles “swell”, changing the way they interact with light and finally giving off a different color. This interaction involves optical phenomena like scattering and interference. A representation of this phenomenon is shown below. It should be noted that the change in coloration shown in the figure is purely for showcasing the effect, and does not aim to be scientifically precise. (7,8,9)

This shift in coloration is a result of the material’s nanostructure, and not pigments. Thus, it can be described as a structural color, reminding us of iGEM Athens 2020’s project, “Morphae”, whose objective was the production of structural color using bacteria. (10)

This color is also non-iridescent (not affected by the viewing angle) and can then be captured and quantified by a camera. The resulting data could then be analyzed using various statistical methods.

Project

So, what can we do?

Our project can be condensed into the following steps:

Discover an M13 pVIII modification that would make it interact with our target VOC. (See Model)

Genetically engineer the M13 bacteriophage so as to incorporate the determined pVIII modification. (See Design, Engineering)

Produce a solution of the engineered M13 in the appropriate concentration and deposit it onto a suitable substrate. (See Hardware)

Our goal in this competition is not to create a functional diagnostic kit for the early detection of Parkinson’s Disease in the span of a few months. This would require years of basic, translational and clinical research and population studies, along with vast resources and expertise. What we aim for is to use this novel technique which, as far as we know of, has not been used for Parkinson's Disease, and make strides towards its possible implementation in the real world. We are also introducing this technology into the iGEM competition and providing future teams with valuable parts, information and inspiration on its many possible and exciting applications in the field of synthetic biology. To discover more about our journey, keep exploring!