One of the major hurdles in synthetic biology has been the construction of bio-sensors that are accurate over a large scale and that can be monitored without the aid of sophisticated microscopes. Getting a bacteria to signal in one way or another when it encounters an environmental toxin (or something else) is one thing but getting a colony of bacterial cells to signal together in synchrony is a whole lot more difficult. Orchestrating thousands of colonies of bacterial cell is an entirely different ball-game. Yet this is exactly what a group of scientists from the University of California, San Diego have achieved. They report their findings in an article published in the Advanced Online Publications section of Nature. And this is the article that I want to briefly review over here.
To synchronise the bacterial cells within a colony they tapped into a natural mechanism used by bacteria to communicate among themselves, quorum sensing. However, quorum sensing is only effective over a range of a few micrometers. So they used a different communication molecule, one that is in the gaseous phase and hence can diffuse quickly to longer distances. But gaseous molecules are weak and short-lived, being in the vapour phase, they disperse a lot quicker. However when these two communicating mechanisms were synergistically coupled, the results were quite surprising.
To achieve the intracellular coupling they used quorum sensing involving acyl-homoserine lactone (AHL). The principle here is quite simple, each bacterial cell synthesises a certain amount of AHL which acts as an inducer. When this inducer binds with a receptor it causes the expression of certain genes which among other products also include the genes for synthesizing the inducer itself. Thus the inducer is technically inducing its own production, rendering it an autoinducer. However the amount of this inducer molecule, AHL in this case, produced by any bacterial cell is not enough to initiate this positive feedback loop. It is only when that the colony reaches a certain size and when a certain critical concentration of the autoinducer has been achieved, that the expression of the downstream proteins occur. As a result, if you insert fluorescent proteins among them, you get a colony of bacterial cells fluorescing in unison. But mind you, this only gets you a coordinated bacterial colony over a very small range. You need something else to scale this up.
So they put a copy of a gene coding for NADH dehydrogenase ll (ndh) under the control of another lux promoter. Now NADH dehydrogenase II is a respiratory enzyme that produces low levels of H2O2 and superoxide. Now H2O2 being a in the vapour state is able to pass between the 25 micron thick PDMS walls that is used to separate the colonies. This H2O2 , being a reactive oxygen species, initiates a defence mechanism in bacterial cells once it enters them. Among the different global regulatory systems that mediate this defence mechanism is one called the ArcAB system, which (lo and behold!) has a binding site in the lux promoter region. Under normal conditions, ArcAB is partially active, rendering the lux promoter partially repressed. But once H2O2 enters the cell, ArcAB swings into action, thus activating the lux promoter and hence initiating the same quorum sensing mediated communication system and hence synchronising this new colony. Now the researchers did a lot of tests to confirm that it was the external H2O2 , and not endogenously produced molecules, which brought about the desired effects. Describing them is out of scope here but the results confirmed their hypothesis.
So what it is that they finally achieved? Synchronisation of approximately 2.5 million cells over a distance of about 5 mm, exhibiting consistent oscillation with a temporal accuracy of 2 minute compared to the 5-10 minute accuracy of a single oscillator!
The paper includes a bit more about the construction of an arsenic detecting biosensor using this technique but I don’t think that’s entirely relevant here. For a brief description of the experiment and the potential applications of it, watch the video:
Merry Mythmas and Happy New Year to all our readers!