Team:Glasgow/Project

Figure1: Glasgow iGEM 2014 Concept Outline

Abstract and Outline

Glasgow's 2014 iGEM project involved the creation of a new and potentially very useful tool for synthetic biologists. We created a system that, in the presence of a specific stimulus, will switch between expressions of one set of genes and another – a change that will persist in subsequent generations unless reversed. This switch based on a site specific recombinase(φC31 integrase) isolated from the Streptomyces phage φC31. In our system, 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 the sugar arabinose being used as the trigger for our proof of concept.

As a potential application of the switch, we investigated the use of Gas Vesicles – gas filled intracellular structures used by cyano and halo bacteria to regulate their density and float up to more desirable conditions in the water columns. The intention was for these genes to be turned ON by our switch. The genes being switched off are crucial motility 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 flotation. This change in behaviour, regulated in a cell autonomous and heritable fashion, would allow for efficient harvesting of cells in a large variety of biotechnology applications. While we created biobricks for the required genes, and were able to disable and restore 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 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 the main structural components of gas vesicles GvpA and GvpC, thus 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 restore their function in a controlled fashion (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 – Photosynthetic bacteria would be engineered to take up high concentrations of intracellular salt from sea or brackish water. The stimulus for the switch would be salt concentration. Once a high enough concentration was reached, the cells would switch off their motility genes, switch on expression of gas vesicle genes and float up to the surface for easy collection – removing salt from the water. 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.