How to control GMOs with molecular ‘lock and key’

June 18, 2015

Researchers achieve biocontainment of genetically modified organisms (E.coli in this model), using mutated sites in a protein (partially shown here) as a lock (red areas) to keep the E.coli inactive until multiple molecular copies  of the key (the chemical benzothiazole in this case) are fitted in the specific red pockets. (credit: Gabriel Lopez, UC Berkeley)

UC Berkeley researchers have developed a low-cost, easy method of biocontainment of bacteria to contain accidental spread of genetically modified organisms (GMOs). The used a series of lock-and-key genetic mutations (in addition to the GMO mutations) that render the microbe inactive unless the right molecule (the key) is added to to the expressed protein to enable its viability.

The work appears this week in the journal ACS Synthetic Biology. It could help deal with recent advances in synthetic biology and genetic engineering of newly created organisms, according to senior author J. Christopher Anderson, an associate professor of bioengineering.

The researchers worked with a strain of E.coli commonly used in research labs, targeting five genes that are required for the organism to survive and devising easy ways to modify them. They created mutations in the genes that would require the addition of the molecule benzothiazole to the expressed proteins to function.

The lock-and-key method

“This approach is very robust and simple in that it only requires a few mutations in the genome,” said Anderson. “The molecule serves as the key, and we engineer the lock.”

“Of the 4,000 genes in E.coli, about 300 are essential to its survival,” notes study lead author Gabriel Lopez, a postdoctoral researcher in Anderson’s lab who started the project as a UC Berkeley graduate student in bioengineering.

The method turns the bacteria into a synthetic auxotroph, an organism that requires a particular compound for its growth (the key). According to the researchers, the method could, for example, potentially be applied to organisms being engineered as pharmaceuticals to treat diseases. That involves introducing foreign organisms into the body, so mechanisms are needed to ensure that the organism is activated only when needed.

Lopez said the researchers added redundancy in the system by adding several lock-and-key combinations into one organism. Switching on a single gene improved viability 100 millionfold, while combining several gene locks resulted in a 10 billionfold increase in viability, according to the study.

The experiment with E.Coli is “just one instance of how you can do it, but my hope is that people will see this and realize we can use this lock-and-key approach with other proteins and other organisms,” said Lopez.

Weakness of a “kill switch”

The researchers noted that other approaches to biocontainment rely on a “kill switch” that poisons an organism if an attempt to use it inappropriately is made. The problem with that, they said, is that the organism may evolve a way to survive that signal.

They noted that in their new approach, the default state of their organism is just the opposite: death. So someone attempting to activate it must actively turn on the genes to enable its survival.

The researchers distinguished their approach as fast, cheap, and easy to deploy, having been demonstrated in an industrially relevant organism.

The inspiration for the development came from techniques dating back to the 1940s, when early biochemists were researching the conditions that would enable microbes to live or die. Biologists discovered that mutating the genes of mold could alter their sensitivity to pH levels, temperature, and other conditions.

“I took those early tools used to figure out how biology works and applied those techniques for making biology work when, where and how we want,” said Lopez.

Not a solution for rogue biohackers

The researchers cautioned that there is no one-size-fits-all solution to biocontainment. “We have to look at what it is that we’re worried about with which application in determining which biocontainment approach is relevant,” said Anderson.

The lock-and-key approach, for instance, is not a practical answer for containing engineered DNA sequences from horizontal gene transfer, a situation in which genetic material could accidentally move from one organism to another, Anderson said.

In addition, this technique is meant to prevent the accidental spread of an engineered organism, but it is not foolproof against intentional attempts to thwart the biosafety net.

“We’re never going to be able to solve the problem of a human actively hacking away at our safety systems,” said Lopez. “Even if we could beat a human today, there will be better equipment and more knowledge 10 years from now, and that will be very hard to secure against.”

The National Science Foundation and the Defense Advanced Research Projects Agency helped support this research.


Abstract of Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21(DE3) Biosafety Strain

Synthetic auxotrophs are organisms engineered to require the presence of a particular molecule for viability. They have potential applications in biocontainment and enzyme engineering. We show that these organisms can be generated by engineering ligand-dependence into essential genes. We demonstrate a method for generating a Synthetic auxotroph based on a Ligand-Dependent Essential gene (SLiDE) using 5 essential genes as test cases: pheSdnaNtyrSmetG, and adk. We show that a single SLiDE strain can have a 1 × 108-fold increase in viability when chemically complemented with the ligand benzothiazole. The optimized SLiDE strain engineering protocol required less than 1 week and $100 USD. We combined multiple SLiDE strain alleles into the industrial Escherichia coli strain BL21(DE3), yielding an organism that exceeds the biosafety criteria with an escape frequency below the limit of detection of 3 × 10–11.