Bioengineers design long-lasting genetic circuitry for cancer-fighting bacteria using a ‘rock-paper-scissors’ strategy

Over roughly the past twenty years, scientists have been trying to harness the power of nature to solve demanding global health, environmental, and manufacturing problems using synthetic biological circuits — living components inside a cell designed to perform logical functions mimicking those observed in electronic circuits.

But, the functionality of present-day man-made biological circuits inevitably crashes in a matter of days to weeks. This is caused by accumulating damage to the organic computer chips, a major obstacle to applying synthetic biology to worldwide crises and conundrums.

Borrowing from how ecologists view relationships of population size changes in an environmental system, researchers have created bacterial strains that synergistically keep each other’s population size and utility in check with synthetic biological circuits that kill off bacteria with unwanted mutated versions.

In an article published in Science, bioengineers from the University of San Diego California (UCSD) designed a community-level system that perpetually protects the functionality of three engineered bacterial strains. The strains in this band of bacterial strains are caught in an ongoing game of ‘rock-paper-scissors’ — a ‘rock’ strain can kill the ‘scissors’ strain but can be killed by the ‘paper’ strain.

The researchers show that this artificial system can be applied, theoretically, to deploy microbes in therapeutics, such as drug delivery vehicles to diseases like cancer. This engineered rock-paper-scissors communal dynamic has major implications for improving synthetic biology to overcome major challenges to humanity, the environment, and all life on earth.

This is a still image pulled from UC San Diego video from three-strain co-culture experiments of E. coli.
This is a still image pulled from UC San Diego video from three-strain co-culture experiments of E. coli.
Fluorescent synthetic bacteria compete in tiny traps (Credit: University of California San Diego / Michael Liao)

Imagine a future where algae soak up pollutants and toxins from rivers and streams, where yeast take in waste and pump out eco-friendly fuel, and where viruses patrol our bodies for cancer or repair failing brain cells. This is the future world imagined by synthetic biology — the scientific field of redesigning and reengineering lifeforms with innovative abilities.

Synthetic biology researchers are tasked with figuring out how to impart instructions in the same way that nature directs hereditary blueprints for how all living things come to be and exist however for a manufactured purpose, such as bioremediation — using lifeforms to consume and break down environmental pollutants.

A common challenge holding back most synthetic biology applications is the high mutation rates for these engineered circuits. Typically working with single-cell lifeforms, like bacteria, viruses, and yeast, the synthetic genetic circuits in these speedy reproducers have high mutation rates.

What happens here is that, for the engineered bacteria to survive, they begin to accumulate mutations that promote their survival at the expense of their programmed function. For this reason, there is a need for strategies to protect engineered bacterial populations.

“As synthetic biologists, our goal is to develop gene circuits that will enable us to harness microorganisms for a wide range of applications,” said Michael Liao, a UCSD bioengineering Ph.D. student and the first author on the Science paper.

“However, the reality today is that the gene circuits we insert into microbes are prone to fail…Whether it be days, weeks, or months, even with the best circuit-stabilization approaches, it’s just a matter of time. And once you lose functionality in your genetic circuit, there is nothing to do but start over.”

This problem has been approached by trying to shield a pure strain of single-cell lifeforms from acquiring mutations that tarnish the living computer chips. In this manner, bioengineers have tried integrating lab-made biological stabilizing elements into single-cell host lifeforms that protect synthetic biological circuits against mutation.

However, although these reinforcement strategies can prolong the march to mutation, the artificial lifeforms inevitably succumb to mutations that increase their survival at the expense of the synthesized circuit’s functionality.

To bring us one step closer to the world as dreamt up by synthetic biology, a method must be developed to extend the life of engineered biological circuits used to instruct single-cell lifeforms to carry out tasks for man-made purposes.

Geisha playing kitsune-ken, an early Japanese rock-paper-scissor or sansukumi-ken game.
Geisha playing kitsune-ken, an early Japanese rock-paper-scissor or sansukumi-ken game.
Geisha playing kitsune-ken, an early Japanese rock-paper-scissor or sansukumi-ken game (c. 1820)

In ecology, interactions refer to how thousands of similar species can coexist in a single ecosystem. Ecological interactions can describe how groups of species — whether predators and prey, hosts and parasites, plants and animals — all affect one another in a single community.

According to classical ecology, when two species compete for the same resource, eventually the more successful species will win out while the other will go extinct. But, if there are three or more competitors, you can prove that many of these species can, theoretically, co-exist forever.

In simple terms, this phenomenon of three or more competitors coexisting in an environment can be understood through the principles of rock-paper-scissors. This childhood game, which originated from Asia (i.e. sansukumi-ken’), is an example of an ‘intransitive’ competition — participants cannot be simply ordered from best to worst.

When placed in pairs, winners and losers emerge: rock beats scissors, paper beats rock, and scissors beat paper. But when all three strategies compete, a deadlock is reached where no one is the undisputed champion of the environment — hence, all participants thrive together.

Interestingly, the rock-paper-scissors model may be common in many ecosystems, such as coral reefs where scientists first observed the dynamic.

Long before humans existed, side-blotched lizards were finding mates using the rules of the human hand game. In the competition for lizard females, three alternative male strategies identifiable by throat color have been locked in an ecological “perpetual motion machine” from which there appears little escape.

More specifically, the ultra-dominant polygynous — practitioners who maintain many female partners — orange-throated males win over the more monogamous mate guarding blue-throated males. The oranges are in turn bested by a sneaker strategy of yellow-throated males. And the sneaker strategy of yellows is, in turn, beat by a mate-guarding strategy of blues to complete the rock-paper-scissors cycle.

Perhaps most relevant to synthetic biology, some bacteria competing in the intestines of mice have been shown to exhibit a rock-paper-scissors dynamic when they engage in antibiotic production. In theory, antibody-producing strains cannot coexist with sensitive or resistant strains in a well-mixed culture, yet all three strains survive together.

Here, strains that produce antibiotics kill sensitive strains; these sensitives outcompete resistant strains, which outcompete the antibiotic-producing strain. The complete rock-paper-scissors system of these three strains results in a state of near balance between continuing and co-occurring processes.

A blue-throated side-blotched lizard

Along these lines, researchers, using the lens of ecology, showed how a small bacterial community can be engineered to stabilize the functionality of a synthetic biological circuit. They created a system of E. coli strains designed to be lethal and immune to one other strain in a three-strain bacterial battle.

So, rather than following the typical approach and placing elements reinforcing the circuits within a single strain, the researchers split up the parts amongst different bacterial subpopulations.

This engineered rock-paper-scissors framework of predictable interactions between bacterial subpopulations enables precise control over the dynamics of the E. coli community. This made it so that a runaway mutation in one E. coli strain would not cause the stabilizing components to fail.

Liao says, “Our work shows there is another path forward, not just in theory, but in practice. We’ve shown that it’s possible to keep circuit-busting mutations at bay. We found a way to keep hitting reset on the mutation clock.”

What’s more, this rock-paper-scissors approach can be used to avoid mutations that occur when microorganisms are instructed to produce and deliver therapeutics for diseases. A critical next step will be to demonstrate that this technique works in live animals.

“We are converging on an extremely stable drug delivery platform with wide applicability for bacterial therapies,” said UCSD bioengineering and biology professor Jeff Hasty, the corresponding author on the study. “There is still work to do, but we’re showing that we can swap populations and keep the circuit running.”

This formula may enable scientists to synthesize biological systems that can be maintained long-term, affecting applications ranging from medical sensors and therapeutics to manufacturing and bioremediation.

Jonathan D. Grinstein, PhD

Written by

Science writer reporting on brains, genes, and biotechnology.

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