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Team:Aix-Marseille/Project
From 2014.igem.org
S.E.colization : Green light, GO!
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.
Project Details
Serine, a signal molecule
Modified E.coli strains we used for this project are mutated for:
- SdaB gene encoding for a Serine transporter
- SdaC gene encoding for a protein able to degrade Serine into 3-PG.
- CheA gene merged with (SH3)4 : we made sure to get rid of any endogenous and exogenous wild type CheA that could not be able to interact with CusR-LZa or phosphorylated CusR-LZa , leading to a decrease in CheA-SH3 intracellular concentration.
- SerA gene: mutated SerA cannot enter the cell anymore to follow its degradation pathway, leading to a continuous increase of the extracellular Serine concentration. Mutated SerA conserves its enzymatic function.
SerA is controlled by CusR promoter which is activated by the variation of extracellular Serine concentration.
Here, the aim is to synthetize the Serine without interruption. When intracellular Serine concentration is high, the protein goes to the extracellular milieu.
The evolution of Serine concentration stimulates or inhibits the cellular division in our system.
CheA‐SH3, convoluted chemotaxy mecanism
Here, a change in the extracellular Serine concentration is detected by Tsr receptor leading to its activation. Then Tsr inhibits the constitutive synthesis of CheA (Histidine kinase). Consequently, CusR transcription factor cannot be activated by phosphorylation anymore.
When extracellular Serine concentration is stabilized, Tsr is not stimulated anymore and stop inhibiting CheA. As a consequence, CheA becomes activated again and phosphorylates CusR.
Note that CheA-(SH3)4 and CusR-LZa are fusion proteins. SH3-pep-LZa is the linker between the two protein complexes.
Regulation of ppGpp intracellular concentration; green light, go !
In the end, the CusR transcription factor activates RelA-RFP expression. RelA is a factor implicated in the increase of ppGpp intracellular concentration. Thus ppGpp blocks the cellular cycle in S phase, leading to the stoppage of E.coli cellular division. In the mean time, CusR fixes to a specific site near from Mesh1 constitutive promoter leading to the inhibition of Mesh1. At this point, bacteria are in the stationary phase, and will turn red.
As we said before, when extracellular Serine concentration increases, CusR won’t be activated. Consequently, RelA-GFP gene won’t be expressed whereas Mesh1-GFP fusion proteins will be produced. The degradation of ppGpp factor by Mesh1 leads to a decrease of ppGpp intracellular concentration resulting in the resumption of E.coli cellular division. I this case, bacteria will appear green.
As the changes in Serine concentration happens in the whole cellular environment, we’re able then to observe the evolution of the cellular division for all of the bacteria.
Furthermore, using both chemotaxy mechanism and changes in the Serine extracellular concentration we would be able to synchronize cells more efficiently and for a longer period than physical or chemical technics that already exist nowadays.
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