Indian Institute of Technology Madras

DESCRIPTION The main aim of this project is to try and solve the hitherto unanswered problem posed by the Shiga toxin using simple synthetic biology tools at an Undergraduate level. The Shiga toxin is produced by harmful strains of some bacteria like E.coli O157:H7 which exist in the form of biofilms in the gastro-intestinal (GI) tract of some cattle. Shiga gets transmitted to humans through the consumption of raw, uncooked beef or meat, through consumption of unclean water (polluted by contact with the infected cattle) and through consumption of unprocessed milk among other routes of transmission. The toxin is recognized by a globotriaosylceramide (Gb3) receptor (CD77) that is present on the surface of human cells but absent in cattle. Through an extensive review of literature, we have come across a nine-amino acid peptide which is a Gb3 mimic i.e it is analogous in structure to the Gb3 receptor, hence when it binds to the toxin, it neutralizes it. Our project aims at using the anti-Shiga activity of this peptide to sequester and nullify the lethal Shiga toxin. The toxin is produced by bacteria in a colony-dependent manner i.e its production is more significant when the bacteria exists as a biofilm. To inhibit further toxin production, we feel it is important to stop biofilm formation. Hence, our project will aim to use indole-3-acetaldehyde (I3A) as a biofilm inhibitor for E.coli O157:H7 biofilms, and consequently, a toxin inhibitor as well. To summarise, we plan to nullify the Shiga toxin by: Expressing the anti-Shiga peptide in our host cells Engineering the production of I3A in our host cells As we envision it at the moment, this strategy to combat the Shiga toxin can be implemented by feeding the cattle with pellets which contain our engineered bacteria immobilized on the surface. Once these pellets reach the GI tract, the production of I3A and anti-Shiga peptide will be induced by the presence of N-acyl homoserine lactone (AHL) which is a cell-cell signalling molecule involved in biofilm formation. MOTIVATION The significance and motivation of our research lies in the fact that there is no established cure to this problem as of today. There have been many different attempts to curb Shiga toxin production, all falling short in some aspect or the other .The difficulty and challenge of this apparently insurmountable task of finding a practical solution is what inspired us to rack our brains at combating the Shiga toxin using the fresh approach of synthetic biology. Shiga toxigenicity is a condition more common in the Western countries because of the high rates of consumption of beef and meat in these places. Statistics show that up till now, the toxin has claimed the lives of more than a million people and many more have been affected severely. An unsolved problem of such magnitude and repercussions definitely warrants some research dedicated to finding a solution to it. Hence, we feel this research needs to be done. Surprisingly, use of antibiotics against the toxin-producing strains of bacteria has shown to increase the toxin levels, contrary to what one expects. The bacteria, under antibiotic stress, initiate a signalling pathway that leads to overproduction of the toxin. We have proposed a completely novel solution employing the powerful tools of synthetic biology. Basically, we will be using synthetically designed DNA constructs coupled with specific BioBrick parts to effect the controlled production/secretion of certain biological entities which, according to our fundamental idea, will help inhibit the Shiga toxin. These ‘biological entities’ are nothing but the anti-Shiga peptide and I3A (biofilm inhibitor). Standard molecular cloning will be used throughout the project. For engineering the production of I3A, we have designed a polycistronic gene construct comprising of TAA1 (tryptophan transaminase) and ipdc (indole pyruvate decarboxylase) - the two enzymes responsible for converting tryptophan into I3A. For expressing the anti-Shiga peptide, we have designed its DNA sequence by codon optimisation for the nine amino-acids. This DNA sequence will have an attached extracellular export signal (ompF) which will translocate the peptide to the extracellular medium. Both these DNA constructs will be cloned downstream of an AHL-dependent promoter (pLuxR) which acts as a regulator in the production of the peptide as well as I3A. When AHL concentration in the gut is high, indicating that there are a large number of cells forming a biofilm, the I3A production will also increase and it will directly help to inhibit further biofilm formation. Hence, this way, we will try to use the bacterial cell-cell signalling molecule as a weapon to initiate an attack against the same bacteria. The desired output will obviously be a regulated production and secretion of I3A and anti-Shiga peptide.