Vibrio harveyi, a bacterium non-pathogenic to humans, is a primary pathogen for marine animals, including shrimps, lobsters, seahorses, sharks,and sea bass. It can cause eye-lesions, gastro-enteritis, vasculitis, and luminous vibriosis in these animals and in particular, has been a great threat to the shrimp industry.
The main goal of our project is to detect the pathogenic bacterium Vibrio harveyi and design a potential antimicrobial agent for it. To do so, we are going to engineer E.coli to manufacture nitric oxide (NO) which is known to have certain antimicrobial effects.
As a first step, we are going to engineer E. coli to detect the presence of the pathogenic bacterium Vibrio harveyi. This means that we are going to use a mutant E. coli strain with a silenced LuxS gene so that this E. coli cannot synthesize its own quorum sensing molecule Autoinducer-2 (AI-2) but is still capable of detecting the AI-2 secreted by the marine pathogen Vibrio harveyi.
We will then further engineer E. coli so that it has a pathway for synthesizing NO by inserting a bacterial Nitric Oxide Synthase (bNOS) gene from Bacillus subtilis into a vector that is then to be transformed into the E. coli. The expression of this gene will be regulated by T7 promoter which is in turn regulated by T7RPol, which is under the control of lsr promoter.
Lsr genes in E. coli are induced once the repressor LsrR is de-repressed. The signalling molecules AI-2, once phosphorylated, de-represses the LsrR repressor. Thus, this means that when the E. coli detects AI-2 from V. harveyi, there is an uptake of AI-2 molecules which are then phosphorylated to phospho-AI-2. These then activate the lsr promoter which then initiates the synthesis of T7RPol. T7RPol activates the T7 promoter and this initiates the transcription of bNOS.
Once transcribed and translated into NOS, NO, an antimicrobial agent for Vibrio harveyi, will be produced.