Team:Glasgow/Project

Figure1: Glasgow iGEM 2014 Concept Outline

Abstract and Outline

Glasgow's 2014 iGEM project involved the creation of a new and hopefully very useful tool for synthetic biologists. With the aid of a genetic switch we created a system that, in the presence of a given stimulus, will switch between one gene and another – a change that will persist in subsequent generations unless reversed. This switch is a site specific recombinase switch (φC31 integrase) isolated from the Streptomyces phage φC31. It flips a section of DNA containing a promoter, in order to turn off the expression of one gene section in favour of another. The switch was proven to work through the use of GFP and RFP on opposite sides of the switch, with arabinose sugar being the trigger for proof of concept.

As a potential application of the switch, we also investigated the use of Gas Vesicles – gas filled structures used by cyano and halo bacteria to regulate their density and float up to more desirable conditions. The intention was for these to be the genes being turned ON. The genes being switched off are crucial flagella genes, such as motA and fliC. In this way, the behavior of the bacteria would be switched from a random run and tumble mode to a simple upwards floatation. While we created biobricks for the required genes, and were able to disable the swimming behaviour of our bacteria, we were never able to get the bacteria to produce fully-fledged gas vesicles.

We have thus divided the project into 4 main sections. These are:
The Switch
Includes work on both the main switch, the integrase and the proof of concept genes; RFP and GFP.

The Gas Vesicle Proteins
This section describes the work that was done in order to get our E.coli to express GvpA and GvpC, and thus gas vesicles (leading to flotation).

Mobility Proteins
In order to switch the swimming of E.coli on and off, we had to both knock out mobility genes successfully AND reinsert them as we desire (i.e as Biobricks) - with as much recovery of motion as possible.

Dry Lab Modelling and Measurements
We wanted to know how the introduction of gas vesicles would affect a bacteria's swimming, and how they would behave with gas vesicles only. Following on from this, we developed a measuring system that could potentially be used to track the speed of these floating bacteria.

The general public's reaction to our research and its potential uses was gauged through a series of events at the Glasgow Science Centre. We also consulted with other synthetic biology institutions, to ask their opinions of the switch/vesicle system as a useful tool for the field.

Potential Applications

Though the project, due to issues discussed in the relevant sections, eventually began focusing on the switch as a tool for others to customise, we have foreseen a number of viable applications, both using the gas vesicle system outlined above. Two examples are:

• Desalination – the stimulus for the switch would be salt concentration. Once an adquent concentration was reached, the cells would float up to the surface for easy collection. We envision this system as being a lot less energy intensive than existing desalination processes.
• Increasing efficiency of biofactory systems: with a sufficient intracellular concentration of product (biofuel etc) being the trigger for the gas vesicles, “finished” cells would float to the surface ready for harvesting, increasing efficiency.