Synthetic biologists are fitting the genomes of microorganisms with synthetic gene circuits to break down polluting plastics, noninvasively diagnose and treat infections in the human gut, and generate chemicals and nutrition on long-haul space flights.
Although such technologies show great promise in the laboratory, they require control and safety measures to ensure that the engineered microorganisms keep their functional gene circuits intact over many cell divisions and are contained to the specific environments they are designed for.
Past efforts led by Harvard Medical School researchers and Wyss Institute for Biologically Inspired Engineering core faculty members Pamela Silver and James Collins have created kill switches in bacteria that cause them to commit suicide in laboratory conditions when they are not wanted anymore.
Now the Silver team has developed two new kill switches that address these challenges.
These latest-generation switches, described Nov. 16 in Molecular Cell, are self-sufficient and highly stable in bacterial populations that evolve and last over many generations. They ensure that only bacteria with intact synthetic gene circuits survive and confine bacteria to a target environment at 37 Celsius, or 98.6 Fahrenheit, the normal temperature of the human body, while inducing them to die at lower temperatures.
“We needed to take our previous work further and develop kill switches that are stable in the long run and would also be useful in real-world applications,” said Silver, who is the Elliot T. and Onie H. Adams Professor of Biochemistry and Systems Biology at Harvard Medical School.
For the first type of kill switch, called the Essentializer, Silver’s team leveraged their previously engineered memory element that allows E. coli bacteria to remember an encounter with a specific stimulus in their environment. The memory element, derived from a bacteria-infecting virus called bacteriophage lambda, either remains silent or reports the occurrence by permanently turning on a visible reporter transgene that the scientists can trace. The signal can be any molecule, for example, an inflammatory cytokine in the gut or a toxin in the environment.
In their recent study, the team devised a way that ensures the memory element is not lost from the genome during the evolution of the bacterial population over more than a hundred generations. During that time, the genomes of individual bacteria acquire random mutations, which also could potentially occur in the memory element, destroying it in their wake. The researchers introduced the Essentializer as a separate element at another location in the bacterium’s genome. As long as the memory element remains intact, either of two phage factors that control its function also inhibits the expression of a toxin gene encoded by the Essentializer. However, the toxin gene remains somewhat “leaky” and still produces residual amounts of toxin that can kill the cell. To keep those residual toxin levels at bay, the researchers included a second gene in their kill switch, which produces low levels of an antitoxin that can neutralize small amounts of the toxin.
“By tying the function of the memory element to that of the Essentializer, we basically link the survival of E. coli bacteria to the presence of the memory element,” said first author Finn Stirling, an HMS graduate student in systems biology working with Silver. “The removal of the memory element from the bacterial genome, which also eliminates the two toxin-suppressing phage factors, immediately triggers the kill switch to produce high amounts of toxin that overwhelm the antitoxin and eliminate the affected bacteria from the population.”
To create this sophisticated system of checks and balances, the scientists also made sure that the kill switches themselves remained fully intact, which is an important prerequisite for future applications; we verified that they were still functional after about 140 cell divisions.
The second kind of kill switch, which the team dubbed Cryodeath, confines bacteria to a specific temperature range using the same toxin/antitoxin combination but regulating it differently. While again, low levels of the antitoxin were produced, the toxin gene was linked to a regulatory sequence that confers cold sensitivity. Shifting the bacteria from 37 C, where they are supposed to thrive, to 22 C, potently induced expression of the toxin and killed the bacteria.
In a set of proof-of-concept experiments, the team demonstrated the usefulness of Cryodeath in vivo. After introducing an E. coli strain containing the kill switch into mice, only 1 of 100,000 bacteria was viable in fecal samples.
“This advance brings us significantly closer to real-world applications of synthetically engineered microbes in the human body or the environment,” Silver said. “We are now working toward combinations of kill switches that can respond to different environmental stimuli to provide even tighter control.”
“This study shows how our teams are leveraging synthetic biology not only to reprogram microbes to create living cellular devices that can carry out useful functions for medicine and environmental remediation but to do this in a way that is safe for all,” said Wyss founding director Donald Ingber, who is also the HMS Judah Folkman Professor of Vascular Biology at Boston Children’s Hospital and professor of bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.