Team:MIT/Description

Description

Overview

  1. Inspiration

  2. Background

  3. Description

  4. Goals

Inspiration

As our team brainstormed potential project ideas, we converged on a shared interest in the gut microbiome, a rich topic that is connected to various aspects of human health. As we brainstormed ideas, we came across various metabolic disorders, such as phenylketonuria (PKU) and we looked at previous iGEM projects on rare metabolic disorders such as Phenylketonuria (PKU), Wilson's Disease, and Hemochromatosis that sparked our interest. One of our mentors suggested we look into Maple Syrup Urine Disease, and it seemed like a very interesting disease with room to improve treatment plans, as well as a great opportunity to work with gut health and metabolic health.

Background

Typical metabolism of branched chain amino acids (BCAAs) post-absorption occurs by the reversible transamination of BCAAs 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), occurring primarily in the liver. In classical MSUD, BCKDH is non-functional, resulting in the circulatory accumulation of BCAAs and their keto-acid derivatives. MSUD can also 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 consists of strict dietary management to limit protein intake and dialysis, hemofiltration, and/or tube feeding during metabolic decompensation episodes, which can be triggered at any point in the patient’s lifetime by physiological stress, regardless of diet or disease severity. Currently, the only cure for MSUD of any severity is a liver transplant, which reverses all dietary restrictions. However, as with all transplants, waiting time, access to successful transplants, and patient willingness to undergo invasive procedures, especially due to youth or milder forms of MSUD, pose significant barriers to widespread curative measures.

For individuals suffering from MSUD, the ability to control diet is crucial for preventing irreversible neurological damage prior to or in lieu of liver transplantation. Treatments to provide dietary flexibility for young children just past infancy (whose proclivities for sampling by far surpasses the abilities of our team) and individuals waiting for, unable to obtain, or unwilling to undergo transplants are currently lacking. This is the gap in MSUD research that our project aims to address.

Description

Using an activating recombinase switch post-manufacture, our B. subtilis probiotic is engineered to:
1) increase intracellular BCAA concentrations through constitutive expression and increased copy number of bcaP, a high-affinity BCAA importer, and gene knockout of azlC and azlD, both BCAA exporters.
2) increase intracellular BCAA breakdown through constitutive expression and increased copy number 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 live a normal life with a normal diet. Additionally, we see our probiotic being incorporated into baby formula as infants with MSUD may die within several days 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.