Team:UIUC Illinois/Project/Background Info

Cardiovascular Disease
Cardiovascular disease (CVD), also known as heart disease, has been the leading cause of death in the United States for over twenty years and is becoming a severe global health issue. Cardiovascular disease describe a slew of heart-related health problems, including myocardial infarctions, heart failure, and stroke. A major source of these problems is the onset of atherosclerosis, a condition characterized by the buildup of plaque in one’s arteries. There are a variety of causes of atherosclerosis, but our project addresses the proatherogenic substance trimethylamine-n-oxide, a metabolite of certain chemicals often found in red meat and energy drinks.


Intestinal Microbiota Promotes Atherosclerosis
Meat consumption correlates to high risk of cardiovascular disease (CVD), mostly because the high level of fats and cholesterol in diet may promote atherosclerosis. Recent research concluded that those two factors may not be sufficient in establishing the link between meat consumption and CVD risk. Researchers in Cleveland Clinic found that intestinal microbiota (gut bacteria) metabolize dietary L-carnitine into trimethylamine (TMA), which is further converted to trimethylamine-N-oxide (TMAO) and accelerates atherosclerosis in mice.

L-carnitine, a quaternary ammonium compound, plays an essential role in the generation of energy in cells due to its function in transporting fatty acids from the cytosol into the mitochondria. For this reason it is sometimes sold in the form of "food supplements" and is marketed as a "natural" health product. However, for normal healthy adults, the cells of the human body produce sufficient amounts of L-carnitine as needed. Recent research in mice with intact intestinal microbiota suggested long-term carnitine consumption "reduced in-vivo reverse cholesterol transport." They also analyzed 2,595 cases of patients who were undergoing cardiac evaluation, and found out a increased rate of cardiac events among those patients with high plasma L-carnitine levels and high TMAO levels.

[[[[[L-carnitine, a quaternary ammonium compound, is essential for generating energy as transporting fatty acids from the cytosol into the mitochondria. Therefore, it is widely available as "food supplements" and being marketed as "natural health products". However, for normal healthy adults, the cells of the human body produce it as needed. So people start to question, is it really good for our body to consume excessive amount of L-carnitine, do we really need it as a supplement? Researchers analyzed 2,595 cases of patients who were undergoing cardiac evaluation, and found out a increased rate of cardiac events among those patients with high plasma L-carnitine levels and high TMAO levels. In order to investigate the mechanism further, recent research in mice with an intact intestinal microbiota, suggested chronic carnitine consumption "altered cecal microbial composition" and "reduced in vivo reverse cholesterol transport". Moreover, when the gut flora was suppressed by antibiotics, the TMAO level turned out to be normal. An interesting discovery also pointed out that vegans and vegetarians produced less TMAO when they were in the chronic L-carnitine consumption trail. With that being said, a well established link between intestinal microbiota and atherosclerosis was confirmed.]]]]


Current Solutions
Current approaches in treating CVD are invasive and come with long recovery times, therefore it would be ideal to start from prevention. The best way to avoid microbiota-dependent atherosclerosis is through diet control and also exercise. However, self-control alone is not the most effective method for promoting public health. Specific to microbiota dependent atherosclerosis, researchers have reported that multiple bacterial strains in gut have different capacity in metabolizing L-carnitine and produce TMA, therefore it is difficult to target and eliminate them. TMA builds up will lead to fish odor syndrome and cause strong body odor, owing to its release via sweat and breath. Therefore it is not wise to cut the process from TMA to TMAO. And so far there are no research to directly target TMAO. So current suggestion is to inhibit the activity of gut flora by ingesting antibiotic cocktail. However, this method is far from satisfying. Because antibiotic pressure might lead to a selection of "super bacteria", which has multiple resistance to antibiotics and might do harm to our body and can't be killed. Also, the activity of gut flora is considered beneficial to host in various aspects, therefore it is crucial to keep a balance of the bacteria composition.


Intestinal Microbiota and Synthetic Biology
Thousands of gut bacteria reside within the human digestive tract, therefore it is regarded as "an ideal bioreactor within human body". Starting from 2008 to last year, a continues interest in engineering intestinal microbiota has been maintained within synthetic biology and iGEM. In 2008, team Caltech engineered the microbial community and was trying to assist with solving various diseases, such as lactose intolerance. In 2012, more than 5 teams decided to dig deep in solving problems by utilizing "our natural bioreactor". Team Trieste developed a safe probiotic platform to express protein in gut flora. Team SDU-Denmark constructed e.coli to produce prebiotics inulin and was trying to inducing changes in the gut flora therefore lower the risk for obesity. Also hoping for treating obesity, team WHU-China designed devices to metabolize the excessive energy in human intesinte. Similar methods are used for cancer related purposes. Team Penn demonstrated that their drug delivery system can be implemented in ingested probiotics which might promote bacterial based therapies. With a similar purpose, team HKUST-Hong Kong delivered their drug by mofified B.subtilis to cancer cells in the lower digestive tract.


Chassis: Escherichia coli Nissle 1917
Safety is always a major concern when we talked about bacteria based treatment, so we have chosen to work with escherichia coli strain Nissle 1917 as the platform for creating a safe probiotic. Claimed by manufacturer of its commercial form "Mutaflor", Nissle 1917 is "one of the best-examined and therapeutically relevant bacterial strains worldwide". E. coli Nissle 1917 has been widely tested for more than 80 years, mostly as a drug deliver system. The strain has T7 RNA Polymerase, which can transcribes only DNA downstream of a T7 promoter. In 2012, Team Trieste developed a safe probiotic platform based on Nissle 1917 to avoid horizontal gene transfer, which add to its safety.


Our Solution: Cardiobiotics
Based on the finding that chronic ingestion of carnitine-riched food will cause the composition change in gut flora, specifically induced the thrive of L-carnitine loving bacteria, the UIUC-Illinois iGEM Team developed a better strategy to construct a probiotic that will out-compete the current gut flora for L-carnitine. With our genetic parts, our genetically modified Nissle 1917, named as Cardiobiotics, presents a never-before-seen application of synthetic biology to the field of cardiovascular health. This summer, Cardiobiotics was tested effectively uptake L-carnitine and diverted to the pathway leads to 3-dehydrocarnine.