Molecular On/Off Switches in Bacterial Design
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Controlling Synthetic Bacteria
“Kill switches” ensure that genetically engineered bacteria survive only in certain environmental conditions.
Two synthetic gene circuits allow researchers to keep genetically engineered (GE) microbes alive only under specific conditions, and to kill them when their services are no longer needed. The circuits, described today (December 7) in Nature Chemical Biology,could help pave the path to safe diagnostics, therapies, or environmental remediation strategies that rely on GE bacteria.
“This is yet another step forward towards better biosafety and biocontainment based on certain aspects of existing technology,” said Guy-Bart Stan, a synthetic biologist at Imperial College London who was not involved in the study.
Study coauthor James Collins, a synthetic biologist at MIT, began to design these gene circuits, or “kill switches,” after becoming interested in using GE microbes for diagnostic and therapeutic purposes. “We were motivated to begin working on the topic as synthetic biology has moved increasingly toward real-world applications,” Collins told The Scientist. Other groups are working to engineer microbes for bioremediation and industrial processes, among other things.
But with genetic modification comes the concern that scientists will create new and uncontrollable species that outcompete or share their genes with wild-type organisms, permanently altering the environment or endangering people’s health.
Earlier this year, two research teams led by Yale bioengineer Farren Isaacs and Harvard geneticist George Church showed that they could genetically modify Escherichia coli to incorporate synthetic amino acids into essential proteins. When the bacteria are not fed the amino acids, they cannot produce these essential proteins, and so they die. This strategy yields bacteria that are very unlikely to survive without support from scientists but requires intensive engineering of the bacterial genome. (See “GMO ‘Kill Switches,’” The Scientist, January 2015.)
In contrast, Collins and his colleagues set out to create kill switches that could work in a more diverse range of microbes. “Our circuit-based safeguards can be conveniently transferred to different bacterial strains without modifying the target cell’s genome,” he wrote in an email.
First, Collins and his colleagues generated a kill switch called “Deadman,” named for a locomotive braking system in which the train will only run if the engineer is affirmatively holding down a pedal. In the microbial version of Deadman, a researcher must feed bacteria a substance called anhydrotetracycline at all times, or else the microbes will express a toxin and self-destruct.
The researchers generated a genetic circuit containing genes for the proteins LacI and TetR, a toxin that is only expressed in the absence of LacI, and a protease that degrades LacI. Under normal circumstances, TetR is preferentially expressed over LacI. TetR expression also triggers expression of the protease, which degrades any LacI that has been expressed. Without LacI, cells express the toxin and die. But when the cells are fed anhydrotetracycline, TetR is inhibited and LacI is expressed. LacI represses the toxin and keeps the cells alive.
Other versions of the Deadman circuit can be designed to degrade essential proteins in the absence of anhydrotetracycline, said Collins.
A second kill switch, “Passcode,” similarly requires that researchers maintain a specific environment for cells lest they express a toxin. Passcode requires a combination of input molecules for cells to survive. The system relies on hybrid transcription factors, each with one component that recognizes a specific DNA sequence, and one component that is sensitive to specific small molecules, such as galactose or cellobiose. One hybrid transcription factor, factor C, turns off expression of a toxin. Two other hybrid transcription factors, factors A and B, suppress expression of factor C. But specific small molecules can keep them from interacting with C. Another small molecule could prevent C from repressing the toxin. Therefore, to keep the cells alive, researchers must provide them with two small molecules that keep factors A and B in check, and make sure not to give them a third small molecule that will interfere with C.
Scientists designing Passcode kill-switches could make hybrid transcription factors respond to whatever combination of small molecules they desired, said Collins. “The strength of our kill switches lies in their flexibility and their ability to detect complex environmental signals for biocontainment.” He noted that companies hoping to keep others from using their cells could keep the recipe for their feed a secret.
“The great advantage is that you can effectively scale this and create different combinations of environments that contain different cocktails of these small molecules, thereby allowing you to effectively create a suite of cells that are going to be viable in different environments,” said Isaacs.
But Church warned that Collins’s circuit-based approach might not as effectively contain bacteria as an amino acid-based method, like one his group developed, since the cells are not fundamentally dependent on foreign biology to survive.
“If you need to have the ability to really scale your containment across a number of different species, then I could see the Passcode kill switches would be incredibly valuable,” said Isaacs. “If you are very concerned about escape frequencies and your degree of biocontainment, maybe you’d opt for something where the organism has been recoded and it relies on a synthetic amino acid.”
Still, Stan said the new paper is a demonstration that creating easy-to-insert kill switches based on genetic circuits is feasible. “I think what they wanted to show in the paper is basically that using some existing genetic circuitry . . . you can obtain biosafety for the here and now.”
GMO “Kill Switches”
Scientists design bacteria reliant upon synthetic amino acids to contain genetically modified organisms.
One of the biggest concerns about genetically modified organisms (GMOs) is that they can infiltrate wild populations and spread their altered genes among naturally occurring species. In Nature today (January 21), two groups present a new method of containing GMOs: by making some of their essential proteins reliant upon synthetic amino acids not found outside of the laboratory.
“What really makes this a valuable step change is that kill switches beforehand were very susceptible to mutation or other conditions, such as metabolic cross feeding, from basically inactivating them,” said Tom Ellis, a synthetic biologist at Imperial College London who was not involved in the studies. The new approach circumvents some of those problems by making it extremely unlikely for the genetically modified bacteria to be able to survive outside of the conditions dictated by their custom-designed genomes.
Both research teams—one led by George Church at Harvard Medical School and the other by Farren Isaacsat Yale University—based their work on so-called genetically recoded organisms (GROs), bacterial genomes that have had all instances of a particular codon replaced by another. Church and Isaacs, along with their colleagues, had previously developed this concept in collaboration. Since then, their respective groups designed the replacement codons to incorporate a synthetic amino acid, and engineered proteins essential to the organism to rely upon the artificial amino acid for proper function.
“Here, for the first time, we’re showing that we’re able to engineer a dependency on synthetic biochemical building blocks for these proteins,” Isaacs told reporters during a conference call.
Both teams found that the cells perished in environments lacking the synthetic amino acid. Although the technology is not ready for industrial-scale deployment, the scientists suggested that such an approach could be applied as a safeguard against the escape of GMOs.
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‘Deadman’ and ‘Passcode’ microbial kill switches for bacterial containment
Clement T Y Chan, Jeong Wook Lee, D Ewen Cameron, Caleb J Bashor & James J Collins
Nature Chemical Biology(2015) http://dx.doi.org:/10.1038/nchembio.1979
Figure 2: The fail-safe mechanism for Deadman circuit activation.
http://www.nature.com/nchembio/journal/vaop/ncurrent/carousel/nchembio.1979-F2.jpg
To demonstrate active control over host cell viability, cells grown under survival conditions (with ATc) were exposed to 1 mM IPTG to directly induce EcoRI and mf-Lon expression. Cell viability was measured by CFU count and is displayed…
Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the ‘Deadman’ and ‘Passcode’ kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently killEscherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.
Nontoxic antimicrobials that evade drug resistance
Stephen A Davis, Benjamin M Vincent, Matthew M Endo, Luke Whitesell, Karen Marchillo, David R Andes, Susan Lindquist & Martin D Burke
Nature Chemical Biology 2015;11:481–487 http://dx.doi.org:/10.1038/nchembio.1821
Drugs that act more promiscuously provide fewer routes for the emergence of resistant mutants. This benefit, however, often comes at the cost of serious off-target and dose-limiting toxicities. The classic example is the antifungal amphotericin B (AmB), which has evaded resistance for more than half a century. We report markedly less toxic amphotericins that nevertheless evade resistance. They are scalably accessed in just three steps from the natural product, and they bind their target (the fungal sterol ergosterol) with far greater selectivity than AmB. Hence, they are less toxic and far more effective in a mouse model of systemic candidiasis. To our surprise, exhaustive efforts to select for mutants resistant to these more selective compounds revealed that they are just as impervious to resistance as AmB. Thus, highly selective cytocidal action and the evasion of resistance are not mutually exclusive, suggesting practical routes to the discovery of less toxic, resistance-evasive therapies.
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