Team:TPHS San Diego/Description
Breakdown of Trimethylamine via Trimethylamine Dehydrogenase to Minimize Heart Disease Caused by Red Meat Consumption
Project Inspiration
Our team resides in San Diego, California, an area that is globally recognized for its medical presence and initiatives to improve human health. Scripps Hospital, Sharp Hospital, Rady Children's Hospital, and the American Heart Association all have institutes based in San Diego and maintain a strong influence over the medical industry. At our school, in particular, a recent assembly discussed the potential of plant-based or cell-based meat products as a solution to the many health risks that come with consuming large amounts of red meat. This raised concerns among our student body and team, motivating us to look deeper into preventative measures. These two influences in parallel inspired our school’s iGEM team to research alternative ways we could tackle the heart associated ailments that result from red meat consumption using synthetic biology. Thus, we landed upon our project to target TMA due to its significant contribution to heart disease, namely atherosclerosis.
Project Description
The purpose of our project is to target and prevent a compound called trimethylamine N-oxide (TMAO) from being produced in the body. Several recent studies have pointed to a high plasma concentration of TMAO being a direct correlation with an increased risk for heart disease. The team which originally discovered this compound observed that TMAO contributed to the development of atherosclerosis by facilitating the buildup of cholesterol in the arteries. The presence of TMAO in the body is caused by a series of chemical reactions. When an individual first consumes red meat or eggs, two compounds specific to these foods -- choline and L-carnitine -- enter the gut microbiome where bacterial enzymes convert them into trimethylamine (TMA). TMA is then oxidized to form TMAO in the liver. There are several enzymes that can reduce TMA and TMAO into other safer compounds. For instance, Trimethylamine dehydrogenase (TMADH) which reduces TMA into formaldehyde, methane, and a reduced electron-transferring flavoprotein. Formaldehyde, however, is toxic so our method is to create a biological system in E. coli where TMADH and formaldehyde dehydrogenase are both produced to break down TMA and formaldehyde. Doing so, we will eliminate the precursor of TMAO and the toxic by-product, in order to resolve the problem of TMAO build-up in the body its harmful effects.
Background
A condition known as atherosclerosis is one of the leading causes of stroke, cardiac arrest, and heart disease around the world. It develops when cholesterol accumulates in harmful levels in the bloodstream and results in hardened, clogged arteries. One of the causes involves a buildup of a harmful organic chemical called Trimethylamine N-Oxide (TMAO) in the liver. TMAO is formed when two compounds, l-carnitine and choline, are ingested in red meat and eggs. In the small intestine, gut microbes react with these two chemicals to form a substance known as Trimethylamine (TMA). TMA travels through the bloodstream to the liver where it turns into TMAO in a reaction facilitated by the gene FMO3. The buildup of TMAO is what subsequently leads to atherosclerosis.
Experimental Design
The main goal of our experiment is to prove that TMADH and FDH can degrade their respective substrates in a controlled lab setting. Our project has three main parts: phase one involves building and confirming our recombinant plasmids and testing the induction conditions of those recombinants, phase two involves transforming and testing the secretion of our enzymes on various plate assays, and the third and final phase is mutating our genes via error-prone PCR. In doing so, we can not only determine the conditions for mutagenesis, but in future studies, select and repeat this mutation process over several generations and eventually produce a more efficient enzyme.
