What happens when biologists take up photography for a hobby?
The trick to doing this lies in getting bacteria to sense light or rather specifically in this case, getting E.coli to sense light. This isnt a trivial task because it is important maintain a signalling cascade while enabling the organism to posess a different response element.
The choice of the signalling pathway was a two component pathway (two component signalling pathways are those pathways in which there is a signalling element and a response element and the system is regulated by a kinase) The two component pathway in this case was EnvZ- OmpR involved in porin expression in response to osmotic shock.
A chimera between EnvZ and Cph1 (a gene in Synechocystis; a cyanobacterium) was constructed by analyzing potential crossover points between the two genes. The idea is to change the response domain in EnvZ to one which is light sensitive. This is not really as trivial as it seems. To appreciate the difficulty, one must understand the number of potential crossover points possible and what kinds of crossovers actually produce functional chimaeric response elements.
These chimeras were on plasmids which were transformed into EnvZ chromosomal knockouts of E.coli. These E.coli also had an ompC promoter linked to a lacZ reporter. In the osmotic regualtion system, OmpR binds ompC and activates the expression of the downstream genes. In this case, OmpR will activate LacZ reporter expression.
Now the parts are in place, but the story still has some length to go. The light sensitive protein needs an element called phycocyanobilin. Where does this come from? E.coli does not have the ability to produce these compounds. The advantage of a well characterised genome/genetic system really comes to the forefront here. The necessary genes were again take from Synechocystis and inserted into E.coli.
Lets do a recap on what we have and how the means are going to be attained.
1. A chimaeric protein of EnvZ- Cph1 that responds to light
2. ompC promoter- lacZ fusion which responds to OmpR binding
3. a biosynthetic system that produces phycocyanobilin which is a component of the light sensor.
How does the system function?
The sensor system is turned off when exposed to redlight due to the inhibition of autophosphorylation. (Only when the protein is phosphorylated, the response system is active and lacZ expression occurs) We now have a switch system in place that responds to light by means of the two component system.
The art of photography:
When an image is projected onto an agar plate containing these modified E.coli, the regions where there is a shadow have a lacZ which is expressed. The lacZ converts S-gal into a black insoluble compound thus producing a contrast image on the plate.
The moral of the story:
Synthetic biology is a field that is in its nascent stage and this kind of work shows how far one can go with controlling microorganisms. Craig Venter for instance is making headway in trying to synthesise the first synthetic minimal microbe. The control that this allows us to exert on bacterial systems is truly awesome.
'Enginering Escherichia coli to see light' Levskaya, A et. al. Nature 438, 441- 442 (2005)
Other useful links:
EnvZ- OmpR two component signalling pathway
A U Texas news release containing comments by some of the authors
Nature news update (subscription required)