Team:IIT Madras/Project

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On average, the world loses 2 people to cardiovascular complications every minute; Diabetes claims 4 times that number making it the 6th most common cause of death worldwide. India alone contributes 1 in every 25 of the diabetes cases every year. The World Health Organisation recently reported that up to 245 million people suffer from diabetes worldwide. It is estimated that diabetes consumes between 5 to 10% of the world's health care expenditure, and this is expected to go up to 12% in the next 20 years. There is, however, no known cure to this epidemic, and our only hope is to fall back to the old adage, 'Prevention is better than cure'. It has been known for a while that reducing the calories consumed in the form of sugar will help in the prevention of this disease, and is an essential part of living-with the disease. To date, seven different sweetening proteins have been identified and approved for consumption by the FDA. However, these proteins face several issues in production, separation and purification, making them an ideal candidate for further work, particularly focusing on expression and regulation.

We aim to use synthetic biology to engineer pro-biotic lactic acid bacteria used in the production of dairy products like yogurt, buttermilk and curds, to produce Monellin, a heat and pH stable sweetening protein. If we are successful in engineering Lactococcus lactis, a Gram positive bacteria to express and secrete Monellin, we will be able to produce dairy products low in poly-saccharide-based sweeteners, radically reducing the calorific content of these products. In order to be able to control the level of expression in this system, we plan to develop a regulatory system(s) that can be tweaked to suit varied purposes.

We aim to use synthetic biology to engineer pro-biotic lactic acid bacteria to produce Monellin, a heat and pH stable sweetening protein. If we are successful in engineering Lactobacillus lactis, a Gram positive bacteria to express and secrete Monellin, we will be able to produce dairy products low in poly-saccharide-based sweeteners, radically reducing the calorific content of these products. In order to be able to control the level of expression in this system, we plan to develop a regulatory system(s) that simulates a logical AND gate in response to two bioloical inputs. We plan to use the decreasing pH during curdling and the addition of nicin as the two inputs. To achieve the AND gate we will be using the CRE gene in combination with loxP sites. By placing the loxP sites appropriately, we will create an expression system that will produce the Monellin in a window of conditions.

Our idea was to design a system that would enable us to produce non-saccharide sweeteners to sweeten a diary product, like curd. We chose curd since it has a fermentation step in its production process with various lactic acid bacteria. Our idea was to transform one of these bacterial strains with the gene of a protein based sweetener, provide a reliable export mechanism for the protein and design a robust control strategy for the protein's expression. After a little thought we decided that the most ideal control stratagem would be to use chemical signals from the curdling process to trigger the regulatory mechanism.

While researching the possibility of taking up this project, one of the first things we looked at was the various sweetening proteins that could be utilized. After a brief search through scientific literature we came up with the following options,

From these we looked into the size of the protein, the source organism, the state of the mRNA processing, and the expression in bacterial systems, among other things. From these perspectives we found that Monellin would be the ideal candidate for our project. At 98 amino acids long, Monellin is the shortest of all the proteins and has also been studied extensively. Most importantly, a number of mutants of Monellin have been expressed in bacterial systems like E.coli. After shortlisting the protein, we had to look through the various mutants to determine the one we wanted to use.