Team:St Andrews/project/objectives

Project Description

The Problem: Cholera

Cholera (V. cholerae) infects 5 million people per year, worldwide and can kill within hours if left untreated. Like all diarrheal diseases, cholera is particularly deadly in children.

Whilst the standard treatment for cholera is some combination of fluids and antibiotics, there is no cure. Efforts have been made towards an effective cholera vaccine, but as yet this has been unsuccessful.

It is a truth universially acknowledged that prevention is better than cure and therefore to prevent cholera from multiplying and therefore prevent causing severe illness and potential death.

Our aim is to investigate whether the basic science behind preventing cholera could work.

Know thine enemy. Cholera's Strategy: Quorum Sensing

Most cholera cells entering the gastroenteric system are killed off by stomach acid (HCl), but the few that survive, pass through the stomach and into the gut where they attach to the gut wall and multiply.

Initially, the cholera cells are at a low cell density and therefore autoinducer (a self stimulant to procreate) concentration is low, and virulence factors are expressed.

Once high cell density is reached, sufficient toxin (choreatoxin) exists to cause extreme diarrhoea. At this point, the autoinducer concentration is high, and virulence factors are repressed.

The now avirulent cholera cells detach from the gut wall and are flushed out of the body with the water leaving the intestine and once outside the body are then able to infect a new host.

Our idea is to modify E. coli which are able to communicate with cholerae. Put simply, the E.Coli will talk to the cholerae and trick the cholerae into leaving the intestine before any virulence factors are sufficient to cause illness to the host human.

Our engineered Escherichia coli will harmlessly colonise the gut, and in large numbers secrete the cholera autoinducer, CAI-1. This will cause an immediate high autoinducer concentration to be detected by incoming Vibrio cholerae cells which then quickly become avirulent and leave the body naturally with the passage of products of digestion, without causing harm to the human.

The Plan

Biology Lab Work

(1) Adding new functionality to the signalling parts present in the registry by re-engineering the existing LuxR quorum sensing system to create a bistable switch. This will allow us to infer a signalling molecule concentration required to deactivate the system much lower than the concentration required to activate it. We will characterise this system by using a fluorescent protein reporter and measuring fluorescence at different cell densities.

(2) Adding the cholera autoinducer synthase gene CqsA to Escherichia coli so that CAI-1 is secreted. The eventual aim is that the bistable switching system will be used to control CqsA expression, so that the ability to compete with other bacteria in the human gut is not compromised by this metabolic burden.

Modelling'

Generating ordinary differential equations to model quorum sensing in V. cholerae and on modelling more complex problems such as bistability and multiple quorum loops working in tandem of our parts.