Team:Edinburgh/Project

In the spirit of iGEM, our project’s aim is to design new biological systems that will make synthetic biology more accessible and friendly. Our team plans to achieve this by constructing a bio-electric interface, designing new selectable and counterselectable markers and characterising Citrobacter freundii as a new chassis. In addition, we want to research the subject of Open Source and DIY biology in relation to the laboratory part of our project as well as its future potential.

Bioelectric interface: As part of our project we will attempt to create a bioelectric interface - a way to connect biological and electronic systems in a standardised, inducible and quantifiable way. To achieve our goal we will use the MtrCAB genes (cytochromes, proteins that mediate electron transport) from Shewanella oneidensis. We will transform E. coli with these genes along with a ccm gene cluster (cytochrome c maturation proteins) and couple it to an inducible promoter such as Ars or Lac promoter induced by arsenate or lactose/IPTG. As a result, we should be able to obtain a system that would allow us to measure the rate of electron export in response to an input of arsenate or IPTG. We will model the process using Kappa and MATLAB. Possible methods to measure electron export include construction of a microbial fuel cell or use of the ferrozine assay to measure rate of reduction of iron (III) ions to iron (II).

We are also looking at two ways of making genetically modified bacteria safer to release into the environment:
Selectable and counter-selectable markers: Alternative to antibiotic resistance: We are investigating other ways to distinguish between the cells which have taken up the gene in question and those which have not in order to eliminate the need of antibiotic resistance selection. This would reduce the spreading of antibiotic resistance genes if an engineered bacterium were to be released into the environment.

Chassis characterization: Citrobacter freundii. We intend to characterize this 'friendly lemon bacterium' (a member of the gamma-proteobacteria, like Escherichia coli) in order to assess whether it would be a good chassis for cloning and gene expression. The emphasis will be on testing whether BioBricks designed for E. coli are compatible with Citrobacter. We also aim to characterize other criteria which would have to be known in order for researchers to start using it as a novel chassis. In addition to characterizing its growth requirements and BioBrick compatibility, we hope to sequence its genome to gain more insight into its metabolic pathways and novel genes. Finally, we want to assess whether public opinion would favour less known but safer Citrobacter freundii over Escherichia coli, which may have a bad reputation due to its association with disease and sewage and ability to become pathogenic if exposed to wild type strains.