A complex logic circuit made from bacterial genes

The circuit is designed to act as the controller in synthetic bacteria that monitor and modify their environment


October 12, 2012
By Diana Lutz

Just as electronic circuits are made from resistors, capacitors and transistors, biological circuits can be made from genes and regulatory proteins. Engineer Tae Seok Moon’s dream is to design modular “genetic parts” that can be used to build logic controllers inside microbes that will program them to make fuel, clean up pollutants, or kill infectious bacteria or cancerous cells.

By force of habit we tend to assume computers are made of silicon, but there is actually no necessary connection between the machine and the material. All that an engineer needs to do to make a computer is to find a way to build logic gates — the elementary building blocks of digital computers — in whatever material is handy.

So logic gates could theoretically be made of pipes of water, channels for billiard balls or even mazes for soldier crabs.

By comparison Tae Seok Moon’s ambition, which is to build logic gates out of genes, seems eminently practical. As a postdoctoral fellow in the lab of Christopher Voigt, PhD, a synthetic biologist at the Massachusetts Institute of Technology, he recently made the largest gene (or genetic) circuit yet reported.

Moon, PhD, now an assistant professor of energy, environmental and chemical engineering in the School of Engineering & Applied Science at Washington University in St. Louis is the lead author of an article describing the project in the Oct. 7 issue of Nature. Voigt is the senior author.

The tiny circuits constructed from these gene gates and others like them may one day be components of engineered cells that will monitor and respond to their environments.

The number of tasks they could undertake is limited only by evolution and human ingenuity. Janitor bacteria might clean up pollutants, chemical-engineer bacteria pump out biofuels and miniature infection-control bacteria might bustle about killing pathogens.

How to make an AND gate out of genes The basis of modern computers is the logic gate, a device that makes simple comparisons between the bits, the 1s and 0s, in which computers encode information. Each logic gate has multiple inputs and one output. The output of the gate depends on the inputs and the operation the gate performs.

An AND gate, for example, turns on only if all of its inputs are on. An OR gate turns on if any of its inputs are on.

Suggestively, genes are turned on or off when a transcription factor binds to a region of DNA adjacent to the gene called a promotor.

To make an AND gate out of genes, however, Moon had to find a gene whose activation is controlled by at least two molecules, not one. So only if both molecule 1 AND molecule 2 are present will the gene be turned on and translated into protein.

Such a genetic circuit had been identified in Salmonella typhimurium, the bacterium that causes food poisoning. In this circuit, the transcription factor can bind to the promotor of a gene only if a molecule called a chaperone is present. This meant the genetic circuit could form the basis of a two-input AND gate.

The circuit Moon eventually built consisted of four sensors for four different molecules that fed into three two-input AND gates. If all four molecules were present, all three AND gates turned on and the last one produced a reporter protein that fluoresced red, so that the operation of the circuit could be easily monitored.

In the future, Moon says, a synthetic bacterium with this circuit might sense four different cancer indicators and, in the presence of all four, release a tumor-killing factor.

Crosstalk and timing faults There are huge differences, of course, between the floppy molecules that embody biological logic gates and the diodes and transistors that embody electronic ones.

Engineers designing biological circuits worry a great deal about crosstalk, or interference. If a circuit is to work properly, the molecules that make up one gate cannot bind to molecules that are part of another gate.

This is much more of a problem in a biological circuit than in an electronic circuit because the interior of a cell is a kind of soup where molecules mingle freely.

To ensure that there wouldn’t be crosstalk among his AND gates, Moon mined parts for his gates from three different strains of bacteria: Shigella flexneri and Pseudomonas aeruginosa, as well as Salmonella.

Although the parts from the three different strains were already quite dissimilar, he made them even more so by subjecting them to error-prone copying cycles and screening the copies for ones that were even less prone to crosstalk (but still functional).

Another problem Moon faced is that biological circuits, unlike electronic ones, don’t have internal clocks that keep the bits moving through the logic gates in lockstep. If signals progress through layers of gates at different speeds, the output of the entire circuit may be wrong, a problem called a timing fault.

Experiments designed to detect such faults in the synthetic circuit showed that they didn’t occur, probably because the chaperones for one layer of logic gates degrades before the transcription factors for the next layer are generated, and this forces a kind of rhythm on the circuit.

Hijacking a bacterium’s controller “We’re not trying to build a computer out of biological logic gates,” Moon says. “You can’t build a computer this way. Instead we’re trying to make controllers that will allow us to access all the things biological organisms do in simple, programmable ways.”

“I see the cell as a system that consists of a sensor, a controller (the logic circuit), and an actuator,” he says. “This paper covers work on the controller, but eventually the controller’s output will drive an actuator, something that will do work on the cell’s surroundings. “

An synthetic bacterium designed by a friend of Moon’s at Nanyang Technological University in Singapore senses signaling molecules released by the pathogen Pseudomonas aeruginosa. When the molecules reach a high enough concentration, the bacterium generates a toxin and a protein that causes it to burst, releasing the toxin, and killing nearby P. aeruginosa.

“Silicon cannot do that,” Moon says.

NTU-led research probes potential link between cancer and a common chemical in consumer products

study led by a group of Nanyang Technological University (NTU) researchers has found that a chemical commonly used in consumer products can potentially cause cancer.

The chemical, Zinc Oxide, is used to absorb harmful ultra violet light. But when it is turned into nano-sized particles, they are able to enter human cells and may damage the cells’ DNA. This in turn activates a protein called p53, whose duty is to prevent damaged cells from multiplying and becoming cancerous. However, cells that lack p53 or do not produce enough functional p53 may instead develop into cancerous cells when they come into contact with Zinc Oxide nanoparticles.

The study is led by Assistant Professor Joachim Loo, 34, and Assistant Professor Ng Kee Woei, 37, from NTU’s School of Materials Science and Engineering. They worked with Assistant Professor David Leong, 38, from the Department of Chemical and Biomolecular Engineering, National University of Singapore, a joint senior author of this research paper.

The findings suggest that companies may need to reassess the health impact of nano-sized Zinc Oxide particles used in everyday products. More studies are also needed on the use and concentration levels of nanomaterials in consumer products, how often a consumer uses them and in what quantities.

“Currently there is a lack of information about the risks of the nanomaterials used in consumer products and what they can pose to the human body. This study points to the need for further research in this area and we hope to work with the relevant authorities on this,” said Asst Prof Loo.

The groundbreaking research findings were published in this month’s edition of Biomaterials, one of the world’s top journals in the field of biomaterials research. The breakthrough also validated efforts by Asst Prof Loo and Asst Prof Ng to pioneer a research group in the emerging field of nanotoxicology, which is still very much in its infancy throughout the world.

Nanotoxicology studies materials to see if they are toxic or harmful when they are turned into nano-sized particles. This is because nanomaterials usually have very different properties when compared to when the materials are of a larger size.

Asst Prof Ng said the team will carry out further research as the DNA damage brought about by nano-sized Zinc Oxide particles is currently a result of an unknown mechanism. But what is clear is that besides causing DNA damage, nanoparticles can also cause other harmful effects when used in high doses.

“From our studies, we found that nanoparticles can also increase stress levels in cells, cause inflammation or simply kill cells,” said Asst Prof Ng who added that apart from finding out the cellular mechanism, more focused research is also expected to ascertain the physiological effects and damage that nano-sized Zinc Oxide particles can cause.

Asst Prof Loo pointed out that besides enhancing the understanding of the potential risks of using nanomaterials, advancements in nanotoxicology research will also help scientists put nanomaterials to good use in biomedical applications.

For example, although killing cells in our bodies is typically undesirable, this becomes a positive outcome if it can be effectively directed towards cancer cells in the body. At the same time, the team is also studying how nanomaterials can be “re-designed” to pose a lesser risk to humans, yet still possess the desired beneficial properties.

This research discovery is one of the latest in a series of biomedical breakthroughs by NTU in healthcare. Future healthcare is one of NTU’s Five Peaks of Excellence with which the university aims to make its mark globally under the NTU 2015 five-year strategic plan. The other four peaks are sustainable earth, new media, the best of the East and West, and innovation.

Moving forward, the team hopes to work with existing and new collaborative partners, within and outside of Singapore, to orchestrate a more concerted effort towards the advancement of the fledgling field of nanotoxicology here, with the aim of helping regulatory bodies in Singapore formulate guidelines to protect consumer interests.

The research team would also like to work with the European Union to uncover the risks involving nanomaterials and how these materials should be regulated before they are made commercially available. Asst Prof Joachim Loo, who received his Bachelor and Doctorate degrees from NTU, was the only Singaporean representative in a recent nanotechnology workshop held in Europe. At the workshop, it was agreed that research collaborations in nanotoxicology between EU and South-east Asia should be