Living Machines and Flashing Lights

Researchers at UC San Diego have created living 'neon signs' from glowing bacteria which flash on and off in unison. As described in this week's Nature, the researchers used this technology to create a bacterial sensor for detecting low levels of arsenic.

I like to imagine that the scientists working in Jeff Hasty’s lab at the University of California, San Diego were the sorts of kids who liked to taking their toys apart and reassemble the pieces into something new and better. Because this is the basic idea behind synthetic biology, a relatively new area of science that creates artificial biological systems using building blocks taken from the natural world.

Engineering living organisms as if they are machines is as awesome as it sounds. Current developments put us at the point of having created many of the individual components needed to make entire circuits and systems—individual modules such as switches, counters and clocks. Teaching a cell to count as described by Friedland et al, for example, might sound a little like it has no use in the real world. But what if we could use this to engineer safeguards into genetically-engineered microbes, ensuring that after a certain number of divisions the cell’s self-destruct mechanisms are activated? Putting the pieces together is where the future challenges lie.

One of the more well-publicised examples of synthetic biology is the creation of artificial life in the lab. Rather than wanting the play God at the risk of wiping out the human race, the true goal of this work is to understand existing life by learning what building blocks are required and testing our existing models for how cellular machinery works. But the future of synthetic biology goes beyond blue skies research. Potential uses include creating new plants or microbes that can synthesise foodstuffs or biofuels, pest-resistant crops, or living sensors such as those described in the recent paper published in Nature.

Living sensors make sense—bacteria encounter numerous chemicals, some harmful and some useful, and thus already possess methods for detecting substances in their natural environments. And bacteria are better at doing this than our current chemical detection methods, which often provide single rather than continuous readings and can require calibration each time they are used. The trick is in harnessing these natural systems to create low cost sensors capable of informing us of the presence of poisons, pollutants, or disease-causing organisms.

The paper from Arthur Prindle and Phillip Samayoa et al, describes the development of a sensor capable of detecting arsenic. But this has been a long time coming—Jess Hasty’s lab have been publishing their work in Nature for years, each time building on their earlier discoveries. And it all started with the creation of a simple biological clock in which cells were synchronised to produce blinking light in unison.

It’s been known for years that bacteria are able to talk to their neighbours using a mechanism known as quorum sensing. This involves the release of diffusible chemicals that effectively tell neighbouring cells that they are not alone—only when this signal reaches a critical level do the cells switch on the various genes controlled by this system. One of the prettiest examples of quorum sensing in action is the bacterium Vibrio fisheri’s symbiosis with the Hawaiian bobtail squid. When highly concentrated in the squid, the bacteria are triggered to produce a bioluminescent chemical that would be wasteful to make were the cells free-living in the ocean. The bioluminescence allows the squid to camoflague itself from its prey; the squid in turn provides the bacteria with nutrients.

Bobtail squid from East Timor. The blue glow is a result of Vibrio fischeri luminescence. Photo by Nick Hobgood

This ability of cells to talk to each other was the basis of the biological clock, shown in the diagram below. It involves three genes all under the control of the same promoter—a region of DNA that controls whether a gene is switched on or off. This promoter is controlled by a signalling molecule, AHL, synthesised by the product of the luxI gene. In this way, luxI drives its own expression by creating more AHL which, in turn, induces more luxI expression. As levels of AHL increase, so does the induction of the gfp gene—this makes green fluorescent protein (GFP) which makes the cells glow under UV light. The third gene, aiiA, however, makes a protein which breaks down AHL. Once levels of this inhibitor reach a critical level, AHL levels decrease, all three genes are switched off, and the process starts all over again. This creates oscillations of GFP production with a specific frequency. While the overall amount of GFP can vary depending on growth conditions, this oscillation frequency remains the same because relative amounts of gene expression are kept constant.

Oscillator setup. Figure adapted from paper. AHL concentration drives expression of all three genes. The combination of LuxL making AHL and AiiA breaking down AHL sets up oscillations in AHL concentration.

To turn this simple system into a sensor for arsenic, Jess Hasty’s lab took a promoter capable of detecting levels of arsenite and used it to modulate levels of AHL. They included a second copy of the luxI gene required to make AHL, this one regulated by the concentration of arsenite. When arsenite is present, there is less induction of this second luxI gene. This alters the ratio of luxI:aaiA induction and results in an increase in the period between the GFP oscillations which is correlated with the concentration of arsenic. And we have a sensor!

Increase in oscillation period with the addition of arsenite.

A flashing bacterial colony is pretty, but it’s a long way from a usable sensor. One problem is that one colony of bacteria isn’t going to generate enough GFP to allow detection without expensive and high-tech equipment, plus background noise can also be high. What was needed was many colonies all working together. But AHL diffuses too slowly to synchronise bacteria that aren’t in close proximity to each other.

To solve this problem, the scientists used a microfluidics setup (an example of which I talked about the other day) and a second signalling molecule, H2O2. On a chip approximately the size of a paperclip, 13,000 separate colonies (termed as ‘biopixels’ by the researchers) are induced to oscillate in a synchronised manner.

A Microfludics setup showing comminucation between colonies by H2O2. B Diagram showing the synchronisation of the oscillations by H2O2, which switches on the AHL-controlled promoters to start each oscillation.

The AHL-dependant system was engineered to also be switched on by the presence of H2O2, shown in the figure above. As H2O2 is a vapour, it can travel between colonies of bacteria very quickly. The individual colonies set up their own AHL-dependant oscillations, which produces small bursts of H2O2 that kick starts the synthesis of AHL in neighbouring colonies. In this way, H2O2 acts as a sort of pacemaker, synchronising the production of GFP as shown in the video below. Because everyone likes science when it is pretty, the researchers also used their flashing biopixels to create neon signs. It might sound more like art than science, but this technology has the potential to be turned into a simple hand-held detector for substances such as arsenic within a few years.

Radical Synchronization from UCSD SBL

1. Danino, T., Mondragon-Palomino, O., Tsimring, L. & Hasty, J. A synchronized quorum of genetic clocks. Nature 463, 326–330 (2010)
2. Arthur Prindle, Phillip Samayoa, Ivan Razinkov, Tal Danino, Lev S. Tsimring & Jeff Hasty. A sensing array of radically coupled genetic ‘biopixels’. Nature 481,39–44(2012)
3. Ari E. Friedland1, Timothy K. Lu, Xiao Wang, David Shi, George Church,and James J. Collins. Synthetic Gene Networks That Count. Science 324:5931, 1199-1202 (2009)
4. Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang, Mikkel A. Algire, Gwynedd A. Benders, Michael G. Montague, Li Ma, Monzia M. Moodie, Chuck Merryman, Sanjay Vashee, Radha Krishnakumar, Nacyra Assad-Garcia, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Lei Young, Zhi-Qing Qi, Thomas H. Segall-Shapiro, Christopher H. Calvey, Prashanth P. Parmar, Clyde A. Hutchison III, Hamilton O. Smith and J. Craig Venter. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science 329(5987), 52-56  (2010)