Overall project summary

Our project centers around synchronizing a culture of E.coli cells, so that they all divide synchronously and change color from green to red and back as they go through the cell cycle. More specifically the culture stops dividing full of red cells and then at regular intervals the cells turn green and divide before returning to the red quiescent state.

This project relies on developing several new and original modules and components which we believe will be generally useful to other teams in future projects. The first component is an inducible Serine production system based on a mutant SerA protein that is insensitive to retro-inhibition by the pathway product (serine) allowing the development of bacteria secreting serine. The second component involves re-engineering the chemotaxis system to drive changes in gene expression rather than changes in flagellar rotation direction. This component is particularly innovative and potentially useful as it can form the basis of several different synthetic signaling system allowing regulation of gene expression by a wide range of different signaling molecules. The third component, or set of components, is a series of switches that change the protein expressed in response to a signal, that is rather than a simple induction of expression as observed with most inducible systems, one protein will be expressed while a second is repressed. Again it is hoped that the bricks that make this system will be applicable in multiple new projects. Each of these components is designed so that if they are introduced together they will produce an oscillator that regularly drives the switch modules between their two states. This oscillator will be coupled to color changes, the initiation of the cell cycle, and serine production (to make the feedback loop). A particularity of the oscillator is that by passing through a secreted substrate and sensor system the oscillations should be culture wide, and all the cells should fall into phase. Furthermore by using the chemotaxis system which has an intrinsically differential sensor (rather than an absolute sensor) it is hoped that the oscillations can be driven over many cell cycles.

This project is relatively fundamental but will we hope provide several generally useable and reusable modules for more applied projects where multiple signaling pathways, culture wide oscillators or switches are required. The project involves both experimental constructions, largely derived from previously published work by other authors, resulting in numerous new Biobricks, and also a strong modeling component to understand the function and constraints of the oscillator that we propose to build and the expected effects on bacterial behavior.

Legend

Project Details

Materials and Methods

The Experiments

Results

Data analysis

Conclusions