Team:IIT Madras/Project

• Motivation

• Ideation

This is where the people stuff comes.

Project Motivation

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 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.

Ideation

Our idea went through many levels of modification and rehashing before we were confident enough to state it in its entirety. Here is a brief summary of the entire process, one that took us a significant portion of two weeks.

The Jist

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.

Sweetening Protein

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.

Plan v1.0

To successfully execute our idea it was important for us to come up with a robust control scheme that would be based on the chemical shifts that occur in the fermentation process. We have chosen to consider the pH change and an external input that can be added to the culture. Taking into account the application in curdling we had decided to choose Nisin, a food grade additive to be our second input. We had planned to construct our system in such a way that Nisin would switch ON our expression and the drop of our system below a certain pH would switch OFF the expression. In this way, we were looking to use a fusion promoter (of the type the iGEM 2008 IIT Madras team developed), with the possibility of sensitivity tuners to tune the system to the process. We already knew of the NICE system that was usable in a Nisin-negative strain of L.lactis that we could utilise in our project. We also discovered the presence of a number of promoters under the umbrella of Acid Tolerance Response in Lactic Acid Bacteria. Among these promoters we were looking for a promoter that would function only above a certain pH. During our search we came across the P170 promoter which is can function only in the pH range of 4.5 – 6.5. We believed that our answer lay in the AND fusion of these two promoters. However, soon after speaking to our predecessors and understanding the random dynamics associated with fusion, we dropped this plan, since our system needed a robust and reliably designed control mechanism.

Plan v2.0

Our second idea came up when we heard about a curious gene system known as the CRE-lox system. We came across that CRE-Lox system, originally discovered in mammalian cells, but very effectively transferred to bacterial systems as a recombinase. Specifically, Cre-Lox recombination is a special type of site-specific recombination, which involves the targeting of a specific sequence of DNA and splicing it with the help of an enzyme called Cre recombinase. Cre-Lox recombination is commonly used to generate a conditional knockout, in which a sequence of DNA is only knocked out at a specific time, or on the application of a particular input.
The Cre protein is a site-specific DNA recombinase, that is, it can catalyse the recombination of DNA between specific sites in a DNA molecule. These sites, known as loxP sequences, contain specific binding sites for Cre that surround a directional core sequence which can either be knocked out, or be used as a site for recombination.
Given this new new input, we were able to conceptualize two theoretically possible replacements for the promoter fusion. These ideas rested on the fact that one of the two inputs we utilise as an inducer, needs to only be an impulse for the production of CRE.

Option 1

Heat shock inducer + CRE; Acid tolerance repressor + loxP (Stop) loxP + Sweetener + Secretory tag in pGFP

Application of heat shock casues the (stop) codon to get knocked out, after which the production of the Sweetener is controlled by the pH. But in this case, the sequence of DNA coding for the Sweet Protein still exists and isn't broken down, which could result in problems later on.

Option 2

Acid tolerance inducer + CRE; Nisin inducer + loxP (Sweetener) loxP + Secretory tag in pGFP

The production of the Sweetener is controlled by Nisin, and regulating pH once the Sweetener has reached the desirabe concentration results in the knocking out of the Sequence of DNA coding fro the Sweetener.
This idea gave us an entirely new approach to genetic modification as something that can be reversed after our objective has been accomplished. A bacteria with an external plasmid can function in a bioprocess, at the end of which it can receive a signal which returns it to its wild-type. The only set-back we had during this phase of ideation was that we were unable to find a threshold based pH inducer.

Final Idea

As mentioned above, the Acid Tolerance Response promoter was not as we expected. Unable to find a threshold based device, we used the next best thing that was available to us, a promoter that shows an up-regulation in transcription in a pH window. Also, we discovered that the NICE induction by Nisin could be accomplished only after the culture reached an OD600 of 0.6 since Nisin is an anti-bacterial agent. Based on this, we modified our construct to the following,

Nisin Inducer + CRE; Acid tolerance inducer + loxP (Sweetener) loxP + Secretory tag in pGFP

Here, the Nisin would be the inducer for the CRE which would excise the gene-of-interest when required. In addition the gene-of-interest would be expressed under the ATR promoter, which would operate in the pH window. Our hope was that the pH window matches the curdling window, and the Nisin can be used to eliminate the production at a required time.