Team:Amsterdam//Project/Synthetic biology/Dependecies

Dependent Synechocystis

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Overview

Background

Background summary here

Aim

Engineer an auxotrophic Synechocystis.

Results

Results summary

Parts

List of created parts.

Background

One can construct a synthetic consortium between two species with a variety of methods. Synechocystis could simply be engineered to export a carbon source (such as glucose) for E. coli in a glucose-absent medium. Although such a commensal relationship would definitely under certain conditions be considered stable, E. coli alone is the benefactor - there is no drive for Synechocystis to produce this carbon source. Increased stability is possible by creating a reciprocal relationship between the two species. Therefore, how can Cyanobacteria benefit or depend on E. coli’s presence? Cyanobacteria manufacturers it’s own food and has grown for centuries requiring only sunlight and water, in addition to inorganic elements. In fact, E. coli’s presence actually harms Cyanobacteria by limiting access to precious sunlight. Herein lies this module’s focus: Genetic engineering of an E. Coli dependent Synechocystis for the formation of stable synthetic consortia.

A basic requirement of consortia requires ‘communication’ between the species. Here we will attempt to construct a mutualistic relationship in which survival of one species is obligatory and beneficial for the survival of the other. The mode of communication will be the mutual production and exchange of essential metabolites. Since Synechocystis is an autotroph, one way to realize such a co-dependency is through the engineering of an auxotrophic bacterial strain. E. coli, could then in exchange produce the nutrient Synechocystis has been engineered to require. Ultimately, Synechocystis will produce a carbon source necessary for survival of E. coli, while E. coli produces a nutrient necessary for survival of Synechocystis - an engineered interdependence pathway.

Aim

This module will therefore aim to engineer an auxotrophic Synechocystis. It also aims to test this auxotroph in the presence of an E. coli engineered to produce the nutrient that Synechocystis has been engineered to be deficient in. This is visualized in Figure 1. One might ponder what is the added benefit of this feedback system in our consortia. Indeed, as stated before, stability can be achieved with just Synechocystis feeding E. coli. However, with this feedback loop, we gain numerous benefits over the aforementioned system.

• We consider potential outbreak risks. An E.coli dependent Synechocystis would not be able to survive outside the lab, thus the risk of environmental contamination would diminish.
• An essential part of our project involves the creation of an emulsion based protocol to test potential consortia. If Synechocystis did not require E. coli, testing out the most effective and stable consortia in this manner would result in simply selecting against the presence of E. coli, as Synechocystis would do whatever it takes to increase it’s own growth rate. By having a feedback loop, we are therefore able to select consortia in which the members work well together.
• Assuming that E. coli could produce Arginine faster than Synechocystis can itself. By knocking out the gene to encode for the enzyme to produce arginine, Synechocystis does not need to waste energy in making these enzymes.