Overview

1. Inspiration

2. Background

3. Description

4. Goals

Inspiration

While brainstorming, our team found a shared interest in the gut microbiome due to its large diversity and varied composition of microbes and its interconnectedness with many other areas of human health. Due to the impact of the gut microbiome on nutrition and homeostasis, we decided that it would be an ideal lens through which to look at metabolic disorders. We looked at previous iGEM projects on rare metabolic disorders such as Phenylketonuria (PKU), Wilson's Disease, and Hemochromatosis that sparked our interest. We ultimately settled on Maple Syrup Urine Disease at the suggesion of one of our mentors due to its interesting pathophysiology and the presence of an unmet need due to a lack of an accessible and long-term treatment for the disease.

Background

In healthy individuals, branched chain amino acids (BCAAs) are metabolized through reverse transamination into their corresponding branched chain keto-acids (BCKAs) in skeletal muscle tissue, followed by the oxidative catabolism of excess BCKAs by the branched chain keto-acid dehydrogenase (BCKDH) complex in the liver. In classical MSUD, one or more subunits of the BCKDH complex is non-functional, resulting in the accumulation of BCAAs and their keto-acid derivatives to toxic levels. Depending on the exact mutation present, the effect of this can take several forms of severity, ranging from partial or intermittent function to complete dysfunction. If left untreated, MSUD results in progressive neurological damage and ultimately death. Treatment of the disease consists of strict dietary management to limit protein intake throughout the patient's lifetime. Dialysis, hemofiltration, and/or tube feeding may be required during severe episodes of metabolic decompensation, which can be triggered by physiological stress, regardless of diet or disease severity. Currently, the only permanent cure for MSUD is a liver transplant. However, as with all transplants, waiting time, access to a high level of medical care, and post-transplant complications pose significant barriers to widespread curative measures. Such an invasive solution may also not be desirable or accessible to individuals with less severe versions of the disease.

For the majority of individuals suffering from MSUD, the ability to control the diet is crucial for preventing irreversible neurological damage. However, such control may be difficult or costly to manage, and leaves limited room for error. This is the gap in MSUD research that our project aims to address.

Description

Our B. subtilis probiotic is engineered to:
1) Increase intracellular BCAA concentrations through constitutive expression of bcaP, a high-affinity BCAA importer, and knockout of azlC and azlD, both of which are BCAA exporters.
2) Increase intracellular BCAA breakdown through constitutive expression of ilvE and ilvK, responsible for the transamination of BCAAs into BCKAs, and constitutive expression of the bkd operon, responsible for oxidative decarboxylation of BCKAs.

We envision our probiotic acting as a non-invasive and convenient supplement for individuals affected by MSUD to help them achieve a greater flexibility in their diet. Future directions we hope to take include the incorporation of our probiotic into baby formula, as infants with severe MSUD may die within several months of birth if they are not treated.

Why is this a good application of synbio?

Since our bacteria are expressing many large protein complexes, it is critical to ensure the fitness of our organism. With a recombinase switch, we are able to induce constitutive gene expression in the gut when the time is right. With a BCAA biosensor, the bacteria would be able to autonomously turn on gene expression when the concentration of BCAA’s inside is high enough.

Goals

Wet Lab

Baseline goal is to be able to prove that increasing expression of bcaP and ilvE/ilvK and the bkd operon is able to increase uptake and breakdown of BCAAs from the environment. If we have time, we would like to add in regulatory elements such as the recombinase switch to be able to induce constitutive expression when the probiotic is ingested or when the cells have reached sufficient density. If we have even more time, we would like to add in self-regulatory elements such as a BCAA biosensor that is able to turn on expression.

Modeling

Baseline goal is to come up with models for our recombinase switch, gene expression, enzyme kinetics, and BCAA transport, and B. subtilis population growth using ordinary differential equations, that can be used to make a rate law for the disappearance of branched chain amino acids in an in vitro environment.

Human Practices

As MIT has a tradition of annual meetups with other team, our team set a goal to host a gut microbiome meet-up event with other teams. We also set goals to meet with various experts on metabolic diseases and MSUD and talk to scientists at local biotechs working on similar synbio probiotic solutions and other stakeholders personally affected by MSUD.