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What is gain of function research in genetics? – Cosmos Magazine

Its the rumour that wont go away that SARS-CoV-2 was accidentally leaked from a high biosecurity lab in Wuhan, China. The allegation is that the laboratory was conducting gain of function (GOF) research, and that this produced a potent version of coronavirus that led to the pandemic.

This has led to some scepticism and distrust of the field of research and whether it is necessary to conduct experiments using GOF techniques.

Essentially, GOF research is used to learn how viruses gain new functions through mutation and evolution.

A function is simply a property of an organism, such as plants that are more tolerant to drought or disease, or enzymes that evolved to make our bodies work.

The language about GOF has become loaded with negative connotations that associate this work with dangerous or risky research. But like rhetoric about genetic modification, these connections dont represent the diversity of the field or the security precautions that regulate the research. At its core, though, the research does exactly what the name suggests.

GOF research observes these mutations and sees how certain stimuli might affect evolutionary changes and properties of a virus or organism.

However, in our current climate its often spoken about in a much narrower context, as though its specifically about how a virus changes to move more easily between humans, or how viruses become more lethal. This just doesnt represent the full picture of GOF research.

Viruses evolve rapidly thats why there are so many new SARS-CoV-2 variants. GOF seeks to understand why and how these changes occur, and what environmental factors might influence the process.

In a sense, this is a know-your-enemy approach.

Beyond the benefit to fundamental biology research about the nature of viruses and evolution, GOF contributes to three clear areas: pandemic preparedness, vaccine development, and identification of new or potential pathogens.

GOF research can help us understand the rate at which mutations occur, and how many generations may be needed for a virus to change in a way that will require extra precautions in the community, which is information that is fed into epidemiological modelling.

This GOF information helps predict things such as how likely a virus is to become a nasty variant in a certain population size or density, during a certain season, or within a particular period or time. This informs how we react to a pandemic. Beyond this, it also informs how quickly a virus might mutate to overcome vaccines, and provides genetic information that may be useful in vaccine development. Specifically, GOF research can accumulate potential vaccine candidates in a database that can be accessed if an outbreak occurs because of natural evolution.

In turn, this means vaccine development can be sped up exponentially because candidates are already available.

For instance, a report from a 2015 GOF risk-assessment workshop for expert organisations revealed the genomics information from GOF research. This showed that bat-borne, SARS-like coronaviruses had many strains and mutations that had pandemic potential against which countermeasures need to be developed.

This information led to current pandemic responses and vaccine development the pandemic was already predicted because of a thorough understanding of the evolution of coronaviruses.

In another example, GOF experiments about influenza showed that the virus had the potential to be transmitted between different mammals with only a few changes to the genetic code, and has contributed to seasonal flu vaccines.

GOF research is based on observed evolution and changes to DNA or RNA.

The genome is the sum of all the genetic information in an organism. Some of this DNA or RNA is made up of genes, which often hold information on how to make a protein. These proteins perform functions in our body to make everything work.

These genes can naturally change a bit every generation. This happens because, to reproduce, the DNA of the parent must be replicated. The mechanisms that do this arent perfect, so little mistakes can be made when the DNA is copied.

Most of the time, the changes are tiny just a single unit of DNA (called a nucleotide) could be changed, and it may have no effect on the proteins made. At other times, the tiny change of a single nucleotide can make a gene gain a whole new function, which could be beneficial to an organism.

Natural mutations that occur during reproduction are one example of evolution in action.

These changes happen every generation, so organisms that can breed quickly, such as flies, can also evolve quickly as a species.

This process happens in essentially the same way with viruses, except that viruses have RNA instead of DNA and reproduce asexually. They still make proteins, and they still accumulate mutations, but the major difference is that they can reproduce very, very fast they can start reproducing within hours of being born and evolve at an exceptionally rapid rate.

This is why we have identified so many new variants of SARS-CoV-2 since the beginning of 2020. Every time the virus enters a new host, it reproduces rapidly, and mutations occur. Over time these mutations change the properties of the virus itself.

For example, new mutations may end up making the virus more virulent or cause worse symptoms because the proteins have changed their properties.

In these cases, we would say that the mutant strain has gained a function, and this is what GOF research aims to understand.

The viruses in a lab dont have a human host in which to grow, so researchers grow them in Petri dishes or animals instead.

There are two ways of using GOF in a lab: you can observe the virus mutate on its own (without intervention), or you can control small changes through genetic modification.

The first type of use involves putting the virus in different situations to see how it will evolve without intervention or aid.

This video is an example of GOF research with bacteria (not a virus, but the method is similar). The researchers put bacteria onto a giant petri dish with different concentrations of antibiotics. They leave the bacteria and watch how it naturally evolves to overcome the antibiotic.

The new strains of bacteria were able to be genetically sequenced to see what genetic changes had caused them to become antibiotic-resistant. This experiment can show how quickly the bacteria evolve, which can inform when or how often antibiotics are given, and whether there is a high-enough concentration of antibiotic that can halt the speed at which the antibiotic is overcome by resistance.

Similar experiments can be conducted with viruses to see how they might change to overcome human antibodies and other immune system protections.

Read more: What happens in a virology lab?

The second type of use is through small changes using genetic modification. This type of experiment occurs after a lot of other genetic information has already been gathered to identify which nucleotides in virus RNA might particularly contribute to a new function.

After these have been identified, a single or small nucleotide change will be made to the virus to confirm the predictions gained from genomic research. The modified virus will then be placed on a petri dish or inserted into an animal, such as a rabbit or a mouse, to see how the change affects the properties of the virus.

This type of research is done in specialised laboratories that are tightly controlled and heavily regulated under biosecurity laws that involve containment and decontamination processes.

Read more: How are dangerous viruses contained in Australia?

While the benefits of virus GOF research centre around pandemic preparedness, concerns have been raised about whether the research is ethical or safe.

In 2005, researchers used this technique for viruses when they reconstructed influenza (H1N1) from samples taken in 1918. The aim was to learn more about the properties of influenza and future pandemics, as influenza still circulates, but the controversial study sparked heavy debate about whether it should be acceptable.

The two major concerns are about whether this poses any threat to public health if a virus escapes the lab, or whether the techniques could be used for nefarious purposes.

In the past year, 16 years after the H1N1 study, there has been debate about whether SARS-CoV-2 had spontaneous zoonotic origins, or whether it was created in a lab in GOF experiments, and then escaped.

So now, 16 years after the first controversial H1N1 study, this speculation has pushed GOF research back into the public eye and led to many criticisms of the research field, and regulation of laboratories that use this technique.

In 2017, the US government lifted bans on GOF pathogen research after the National Institute of Health concluded that the risks of research into influenza and MERS were outweighed by the benefits, and that few posed significant threats to public health.

Following concerns about the origins of SARS-CoV-2, however, the rules surrounding GOF research, risk assessments and disclosure of experiments are now under review again, in order to clarify policy.

Read more: The COVID lab-leak hypothesis: what scientists do and dont know

Beyond this, the speculation has sparked further inquiries into the origin of SARS-CoV-2, although the World Health Organization concluded that viral escape from a laboratory was very unlikely.

Regardless, its never a bad thing to review biosafety, biosecurity and transparency policy as new evidence becomes available, and they have been frequently reviewed throughout history.

As for the concern that a government or private entity might abuse scientific techniques for malevolent purposes, scientists can, and do, support bans on research they deem ethically irresponsible, such as the controversial CRISPR babies.

Ultimately, the parameters around how scientific techniques like GOF are used and by whom is not a scientific question, but one that must be answered by ethicists.

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What is gain of function research in genetics? - Cosmos Magazine

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Could editing the genomes of bats prevent future pandemics? – STAT – STAT

Amid the devastating Covid-19 pandemic, two researchers are proposing a drastic way to stop future pandemics: using a technology called a gene drive to rewrite the DNA of bats to prevent them from becoming infected with coronaviruses.

The scientists aim to block spillover events, in which viruses jump from infected bats to humans one suspected source of the coronavirus that causes Covid. Spillover events are thought to have sparked other coronavirus outbreaks as well, including SARS-1 in the early 2000s and Middle East respiratory syndrome (MERS).




With a very high probability, we are going to see this over and over again, argues entrepreneur and computational geneticist Yaniv Erlich of the Interdisciplinary Center Herzliya in Israel, who is one of two authors of the proposal, titled Preventing COVID-59.


Maybe our kids will not benefit, maybe our grandchildren will benefit, but if this approach works, we could deploy the same strategy against many types of viruses, Erlich told STAT.



Erlich and his co-author, immunologist Daniel Douek at the U.S. National Institute of Allergy and Infectious Diseases, now propose an additional measure: creating a gene drive to render wild horseshoe bats immune to the types of coronavirus infections that are thought to have triggered the SARS, MERS, and Covid-19 pandemics. They shared the proposal Wednesday on the Github publishing and code-sharing platform.

Though there is heated debate about whether the Covid-19 virus originated in a lab, most scientists say the virus is most likely to have originated in wild animals. There is strong evidence, for instance, that horseshoe bats carry the coronavirus that caused the SARS outbreak.

A gene drive is a technique for turbocharging evolution and spreading new traits throughout a species faster than they would spread through natural selection. It involves using a gene editing technology such as CRISPR to modify an organisms genome so that it passes a new trait to its offspring and throughout the species.

The idea of making a gene drive in bats faces such enormous scientific, technical, social, and economic obstacles that scientists interviewed by STAT called it folly, far-fetched, and concerning. Among other objections, they worried about unintended consequences with so radically tampering with nature.

We have other ways of preventing future Covid-19 outbreaks, argued Natalie Kofler, a trained molecular biologist and bioethicist and founder of Editing Nature, a group focused on inclusive decision-making about genetic technologies.


Coming from anyone else, the idea might be laughed off.


Now, Erlich says, its worth thinking about how a gene drive could work in bats.


Its almost like creating a self-propagating vaccine in these bats, Erlich said.


Most of the proposals Ive heard involving gene drives have seemed quite attractive, and this is probably the most attractive, he said.


But its been difficult to engineer effective gene drives in mammals. Developmental geneticist Kim Cooper and her team at the University of California, San Diego, engineered a gene drive that spread a genetic variant through 72% of mouse offspring in her lab. That isnt efficient enough to quickly spread the desired trait in the wild.




Biologists also say that Erlichs proposal is unlikely to work in the wild even if researchers get bat gene drives to work in a lab because bats are incredibly diverse.




Youd have to develop systems for entire bat communities, said evolutionary biologist Liliana Dvalos of Stony Brook University. Its the job of visionaries to come up with creative ideas, but this is a giant blind spot in their thinking.


Further, nobody knows how eliminating coronaviruses might affect bats.

We dont know the implications of wiping out coronaviruses in bat populations, because we dont know how bats have evolved to coexist with these viruses, said virologist Arinjay Banerjee of the Vaccine and Infectious Disease Organization at the University of Saskatchewan in Saskatoon, Canada.

Some scientists, though, welcomed Erlichs proposal, hoping that it will focus attention on what it would take to create successful mammalian gene drive systems.


I would be concerned if this proposal detracted from the need to fund public health infrastructure, said Saah. But with that caveat, he added, I think this proposal could make people think, OK, if we were to use this technology in this animal in this system, what would we need to do? There would need to be a foundation of ethical development, of clear understanding, of social systems and trust, and technology built in a stepwise manner.


In the past, maybe we were blinded a little bit by our belief that we would just be able to increase surveillance and identify these pathogens prior to them spilling over, Kindrachuk said. We now realize that this is going to take a lot of different efforts, so theres an aspect from a research standpoint where we continue to look at things like this, and say, what are the top 5 to 10 things we should invest in.

Erlich acknowledges the obstacles to his proposal, but thinks they arent insurmountable. He thinks the project would require an international investment involving a multidisciplinary consortium.

While we totally agree about the technical complexities, technology advances at exponential rates, Erlich said. Things that are nearly impossible now can be totally reachable within a decade or so.


Lets discuss the idea and think about what we can do to identify a very rigorous and cautious way to test this approach, Erlich said. We dont like to mess with nature, but the current situation is not sustainable.

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Inside the risky bat-virus engineering that links America to Wuhan – MIT Technology Review

For Baric, that research started in the late 1990s. Coronaviruses were then considered low risk, but Barics studies on the genetics that allowed viruses to enter human cells convinced him that some might be just a few mutations away from jumping the species barrier.

That hunch was confirmed in 200203, when SARS broke out in southern China, infecting 8,000 people. As bad as that was, Baric says, we dodged a bullet with SARS. The disease didnt spread from one person to another until about a day after severe symptoms began to appear, making it easier to corral through quarantines and contact tracing. Only 774 people died in that outbreak, but if it had been transmitted as easily as SARS-CoV-2, we would have had a pandemic with a 10% mortality rate, Baric says. Thats how close humanity came.

As tempting as it was to write off SARS as a one-time event, in 2012 MERS emerged and began infecting people in the Middle East. For me personally, that was a wake-up call that the animal reservoirs must have many, many more strains that are poised for cross-species movement, says Baric.

By then, examples of such dangers were already being discovered by Shis team, which had spent years sampling bats in southern China to locate the origin of SARS. The project was part of a global viral surveillance effort spearheaded by the US nonprofit EcoHealth Alliance. The nonprofitwhich has an annual income of over $16 million, more than 90% from government grantshas its office in New York but partners with local research groups in other countries to do field and lab work. The WIV was its crown jewel, and Peter Daszak, president of EcoHealth Alliance, has been a coauthor with Shi on most of her key papers.

By taking thousands of samples from guano, fecal swabs, and bat tissue, and searching those samples for genetic sequences similar to SARS, Shis team began to discover many closely related viruses. In a cave in Yunnan Province in 2011 or 2012, they discovered the two closest, which they named WIV1 and SHC014.

Shi managed to culture WIV1 in her lab from a fecal sample and show that it could directly infect human cells, proving that SARS-like viruses ready to leap straight from bats to humans already lurked in the natural world. This showed, Daszak and Shi argued, that bat coronaviruses were a substantial global threat. Scientists, they said, needed to find them, and study them, before they found us.

Many of the other viruses couldnt be grown, but Barics system provided a way to rapidly test their spikes by engineering them into similar viruses. When the chimera he made using SHC014 proved able to infect human cells in a dish, Daszak told the press that these revelations should move this virus from a candidate emerging pathogen to a clear and present danger.

To others, it was the perfect example of the unnecessary dangers of gain-of-function science. The only impact of this work is the creation, in a lab, of a new, non-natural risk, the Rutgers microbiologist Richard Ebright, a longtime critic of such research, told Nature.

To Baric, the situation was more nuanced. Although his creation might be more dangerous than the original mouse-adapted virus hed used as a backbone, it was still wimpy compared with SARScertainly not the supervirus Senator Paul would later suggest.

In the end, the NIH clampdown never had teeth. It included a clause granting exceptions if head of funding agency determines research is urgently necessary to protect public health or national security. Not only were Barics studies allowed to move forward, but so were all studies that applied for exemptions. The funding restrictions were lifted in 2017 and replaced with a more lenient system.

If the NIH was looking for a scientist to make regulators comfortable with gain-of-function research, Baric was the obvious choice. For years hed insisted on extra safety steps, and he took pains to point these out in his 2015 paper, as if modeling the way forward.

The CDC recognizes four levels of biosafety and recommends which pathogens should be studied at which level. Biosafety level 1 is for nonhazardous organisms and requires virtually no precautions: wear a lab coat and gloves as needed. BSL-2 is for moderately hazardous pathogens that are already endemic in the area, and relatively mild interventions are indicated: close the door, wear eye protection, dispose of waste materials in an autoclave. BSL-3 is where things get serious. Its for pathogens that can cause serious disease through respiratory transmission, such as influenza and SARS, and the associated protocols include multiple barriers to escape. Labs are walled off by two sets of self-closing, locking doors; air is filtered; personnel use full PPE and N95 masks and are under medical surveillance. BSL-4 is for the baddest of the baddies, such as Ebola and Marburg: full moon suits and dedicated air systems are added to the arsenal.

There are no enforceable standards of what you should and shouldnt do. Its up to the individual countries, institutions, and scientists.

In Barics lab, the chimeras were studied at BSL-3, enhanced with additional steps like Tyvek suits, double gloves, and powered-air respirators for all workers. Local first-responder teams participated in regular drills to increase their familiarity with the lab. All workers were monitored for infections, and local hospitals had procedures in place to handle incoming scientists. It was probably one of the safest BSL-3 facilities in the world. That still wasnt enough to prevent a handful of errors over the years: some scientists were even bitten by virus-carrying mice. But no infections resulted.

In 2014, the NIH awarded a five-year, $3.75 million grant to EcoHealth Alliance to study the risk that more bat-borne coronaviruses would emerge in China, using the same kind of techniques Baric had pioneered. Some of that work was to be subcontracted to the Wuhan Institute of Virology.

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Science, industry team up in Italy to zap COVID with laser – New York Post

ROME, July 2 A United Nations-backed scientific research centre hasteamedupwith an Italian tech firm to explore whetherlaserlight can be used to kill coronavirusparticles suspended in the air and help keep indoor spaces safe.

The joint effort between the International Centre for Genetic Engineering and Biotechnology (ICGEB) of Trieste, a city in the north ofItaly, and the nearby Eltech K-Lasercompany, was launched last year as COVID-19 was battering the country.

They created a device that forces air through a sterilization chamber which contains alaserbeam filter that pulverizesviruses and bacteria.

I thoughtlasers were more for a shaman rather than a doctor but I have had to change my mind. The device proved able to kill theviruses in less than 50 milliseconds, said Serena Zacchigna, groupleader for Cardiovascular Biology at the ICGEB.

Healthy indoor environments with a substantially reduced pathogen count are deemed essential for public health in the post COVID-19 crisis, a respiratory infection which has caused more than four million deaths worldwide in barely 18 months.

Zacchigna hookedupwith Italian engineer Francesco Zanata, the founder of Eltech K-Laser, a firm specialised in medicallasers whose products are used by sports stars to treat muscle inflammation and fractures.

Some experts have warned against the possible pitfalls of using light-based technologies to attack thevirusthat causes COVID-19.

A study published by the Journal of Photochemistry & Photobiology in November 2020 highlighted concerns ranging from potential cancer risks to the cost of expensive light sources.

But Zacchigna and Zanata dismissed any health issues, saying thelasernever comes into contact with human skin.

Our device uses nature against nature. It is 100% safe for people and almost fully recyclable, Zanata told Reuters.

The technology, however, does not eliminateviruses and bacteria when they drop from the air onto surfaces or the floor. Nor can it prevent direct contagion when someone who is infected sneezes or talks loudly in the proximity of someone else.

Eltech K-Laserhas received a patent from Italian authorities and is seeking to extend this globally.

The portable version of the invention is some 1.8 metres (5.9 ft) high and weighs about 55 lb. The company said the technology can also be placed within air-conditioning units.

In the meantime, the first potential customers are liningup, including Germanys EcoCare, a service provider of testing and vaccination solutions.

The company aims to license the technology for German and UAE markets, an EcoCare spokesperson said in an email to Reuters.

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Khalifa University researchers complete reference genome study for the UAE – WAM EN

ABU DHABI, 5th July, 2021 (WAM) -- A team of scientists from Khalifa University of Science and Technology has completed a significant local genome study that will contribute to nationwide efforts to build a high-quality, comprehensive reference genome for the UAE population.

The first phase of the study - the description of the first whole genome sequences of UAE nationals - was completed in 2019. Subsequently, in 2020, the researchers completed the second phase which described the nature of the genetic diversity found among UAE nationals. This year, the researchers completed the third phase of the UAE reference genome, which supports a broader understanding of the genome composition of the nation.

Following advancements in DNA sequencing and analysis techniques since renowned scientist Craig Venter and his colleagues published the first whole human genome sequence at the turn of this century, the genome study has become part of a major area of research at Khalifa University.

The Khalifa University scientists recently published a report titled A population-specific Major Allele Reference Genome from the United Arab Emirates population in the international journal, Frontiers in Genetics. The study was authored by Dr. Habiba Alsafar, Associate Professor, Department of Genetics and Molecular Biology, Dr. Andreas Henschel, Associate Professor, Electrical Engineering and Computer Science, with Dr. Gihan Daw Elbait and Dr. Guan Tay, from the Center for Biotechnology.

Dr. Arif Sultan Al Hammadi, Executive Vice-President, Khalifa University, said: "Our researchers have published the first whole genome of a UAE national and have followed it up with this reference genome. This will advance our understanding of the genomes of the UAE population, improving the ability of researchers and clinicians to identify genetic causes of diseases that are common in the UAE and the region. This is a stellar achievement in the field of medicine and healthcare, as this will become a fundamental tool that will advance genome and public health research in the UAE, and contribute to nationwide efforts, being led by the recently formed UAE Genomics Council to incorporate genomics into the healthcare ecosystem of the UAE."

The ethnic composition of the population of a nation contributes to its genetic uniqueness. Consequently, it is important to define national reference genomes of its people to avoid any confounding effects which are linked to the use of reference genomes from other national genome sequencing efforts. A total of 1,028 UAE nationals were recruited for this study, as part of the 1,000 UAE genome project that was conceived by the research team when the Center of Biotechnology was founded in 2015. Of these, 129 samples were selected as individuals that are most representative of the genetic diversity of the UAE for construction of the UAERG.

"Despite achieving this major milestone in a relatively short period of time, our work to improve our understanding of how genes contribute to health continues," said Dr. Alsafar and added, "Our next challenge is to decode the genome data to identify genetic markers that better predict the likelihood of disease."

Precision medicine has the potential to profoundly improve the practice of medicine. The goal is to enable clinicians to quickly, efficiently and accurately predict the most appropriate course of action for a patient; a pre-emptive strike to prevent or delay the onset of disease. However, the practice of precision medicine and personalized healthcare is a complex science as it is influenced by a range of factors such as the environment and the inherent characteristics within an individual. Genetics is an important contributor to this complexity and genome science will play a key role in the rollout of future national health programs.

Since the establishment of the Center for Biotechnology, its primary mission sought to address a gap in knowledge relating to the specific genomic features of the UAE population. In 2018, the BTC team outlined a vision for a National Arab Genome project for the UAE in the Journal of Human Genetics. The aim was to address the deficiency in genome data on the UAE population to improve our understanding of genome variants that are unique to the population of the nation. The team led by eminent geneticist Dr. Alsafar, proceeded with the bold ambition to sequence Emirati nationals to provide a reference upon which clinical decisions can be made.

In 2019, Dr. Alsafar led the team that described the first Whole Genomes Sequences (WGS) of two UAE nationals in Nature Publishing Groups Scientific Report. "It was important to achieve this milestone, as the whole genome sequences provided a starting point for construction of a UAE reference panel which will lead to improvements in the delivery of precision medicine, which we hope will eventually lead to improvements in the quality of life of UAE nationals" said Dr Alsafar.

Despite reporting on the first genome of a UAE national, the Khalifa University team continued to sequence samples provided by UAE nationals for research. In mid-2020, the team followed up the report of the first UAE Whole Genome Sequence with two papers in Frontiers in Genetics. These studies showed that the contemporary population of the UAE arose from gradual admixture through complex and long term interactions between local communities of the area that is now the UAE and the people of neighbouring regions.

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Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells – Science Advances


Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

New safeguards are needed for the deliberate release of engineered microbes into the environment, which has promise for applications in agriculture, environmental remediation, and medicine (1). Genetically encoded biocontainment strategies enable attenuation of engineered live bacteria for diverse biomedical applications (24), including as potential vaccines (510), diagnostics (11), and therapeutics (1215). Auxotrophy, which is the inability of an organism to synthesize a compound needed for its growth, is an existing strategy for containment. However, foundational studies of auxotrophic pathogens demonstrated proliferation in relevant biological fluids (16) and reversion to prototrophy upon serial passaging (17, 18). Modern genome engineering strategies can prevent auxotrophic reversion, and auxotrophy has been a key component of microbial therapies that have reached advanced clinical trials. However, the ability for auxotrophs to access required metabolites within many host microenvironments, and after leaving the host, remains unaddressed. Auxotrophy may not be effective in scenarios where engineered living bacteria encounter metabolites from dead host cells (19) or invade host cells (20). Growth of double auxotrophs is supported in vivo by neoplastic tissue (13). Auxotrophy may also be insufficient for tight control of cell proliferation in environments rich with microbial sources of cross-feeding (21), such as gut, oral, skin, and vaginal microbiomes. Given that most naturally occurring microorganisms are auxotrophs (22), it is also unlikely that auxotrophy will limit the spread of an engineered microbe once it leaves the body and enters the environment.

Synthetic auxotrophy may overcome these hurdles by requiring provision of a synthetic molecule for survival of the engineered bacteria. This strategy was first implemented successfully in Escherichia coli by engineering essential proteins to depend on incorporation of a nonstandard amino acid (nsAA) (23, 24). We previously engineered E. coli strains for dependence on the nsAA biphenylalanine (BipA) by computer-aided redesign of essential enzymes in conjunction with expression of orthogonal translation machinery for BipA incorporation (23). Among several synthetic auxotrophs originally constructed, one strain harbored three redesigned, nsAA-dependent genesadenylate kinase (adk.d6), tyrosyl-tRNA synthetase (tyrS.d8), and BipA-dependent aminoacyl-tRNA synthetasefor aminoacylation of BipA (BipARS.d6). This BipA-dependent strain, dubbed DEP, exhibited undetectable escape throughout 14 days of monitoring at an assay detection limit of 2.2 1012 escapees per colony-forming unit (CFU) (23). Although this strain demonstrates effective biocontainment in 1-liter batch experiments, its precise escape frequency and long-term stability remained unexplored.

Here, we perform the first study of evolutionary stability of a synthetic auxotroph with the aid of automated continuous evolution. Continuous evolution better emulates scenarios where biocontainment may be needed by fostering greater genetic variability within a population. We posited that decreasing BipA concentrations would add selective pressure for adaptation or for escape, either of which would be enlightening. Adaptive laboratory evolution of DEP may improve its fitness in relevant growth contexts, as previously demonstrated for its nonauxotrophic but recoded ancestor, C321.A (25). We report that DEP maintains its inability to grow in the absence of synthetic nutrient, even after three parallel 100-day chemostat trials. In addition, we find evidence of adaptation, with evolved DEP isolates requiring 10-fold lower BipA concentration to achieve optimal growth than ancestral DEP (0.5 M rather than 5 M). We resequence evolved populations and perform allelic reconstruction in ancestral DEP using multiplex automatable genome engineering (MAGE), identifying alleles that partially restore the adaptive phenotype. Last, we advance this technology toward host-microbe coculture applications, demonstrating direct mixed culture of DEP and mammalian cells without the need for physical barriers or complex fluidics.

To perform continuous evolution of E. coli, we constructed custom chemostats for parallelized and automated culturing (Fig. 1A). Our design and construction were based on the eVOLVER system (26), an open-source, do-it-yourself automated culturing platform (figs. S1 to S4). By decreasing BipA concentration over time in our chemostats, we provide an initial mild selection for escape and steadily increase its stringency. This design is analogous to a morbidostat, where a lethal drug is introduced dynamically at sublethal concentrations to study microbial drug resistance (27), but with synthetic auxotrophy providing selective pressure. Our working algorithm for automated adjustment of BipA concentration as a function of turbidity is shown in Fig. 1B, and a representative image of our hardware is shown in Fig. 1C (see also fig. S5).

(A) Illustration of a smart sleeve connected to separate nonpermissive media and biphenylalanine (BipA; structure shown in blue) feed lines for automated adjustment of BipA concentration based on growth rate. Pumps and optics are integrated with Arduino controller hardware and Python software based on the eVOLVER do-it-yourself automated culturing framework. (B) Working algorithm for maintenance of cultures in continuous evolution mode. Criteria for lowering the BipA concentration are based on the difference in time elapsing between OD peaks (tpeak OD). Smaller time elapsed between OD peaks is indicative of higher growth rates, triggering decrease in BipA concentration when below a threshold value. (C) Representative configuration of hardware for parallelized evolution in triplicate, with three empty sleeves shown. Photo credit: Michael Napolitano, Harvard Medical School.

Our long-term culturing experiments featured two phases. The first phase included one chemostat (N = 1) that was inoculated with DEP for an 11-day incubation, with an initial concentration of BipA of 100 M and automated adjustment based on growth rate (Fig. 2A). Because we observed no colony formation when the outgrowth from this population was plated on nonpermissive media, we then began a second phase in replicate. We used our population grown for 11 days to inoculate three chemostats in parallel (N = 3) where BipA supply decreased automatically over the following 90 days from 100 M to nearly 100 nM. One controller provided identical BipA concentrations to all three vials at any given time. To determine whether the decrease in BipA supply was due to escape from dependence on BipA, we periodically performed escape assays. We continued to observe no escape, including when we seeded liter-scale cultures and plated the associated outgrowth on nonpermissive media. Evolved isolates were obtained after this procedure (fig. S6), and their growth was characterized across BipA concentrations (Fig. 2B and fig. S7). At 0.5 to 1 M BipA, we observed growth of all evolved isolates and no growth of the ancestral DEP strain.

(A) Timeline for continuous evolution, with detection limits for escape frequency assays shown in parentheses. (B) Doubling times of progenitor and evolved synthetic auxotrophs as a function of BipA concentration, normalized to the doubling time of DEP at 100 M BipA. Error bars represent the SD across technical triplicates within the same experiment.

To identify the causal alleles contributing to decreased BipA requirement of all three evolved isolates, we performed whole-genome sequencing and mutational analysis. We expected that mutations in auxotrophic markers or orthogonal translation machinery associated with aminoacylation of BipA would be observed. However, no variants were detected in the plasmid-expressed orthogonal translation machinery (aminoacyl-tRNA synthetase and tRNA) reference sequence. Instead, in all three evolved isolates, variants were observed in three nonessential genes, all of which are implicated in molecular transport: acrB, emrD, and trkH (Fig. 3A). AcrB and EmrD are biochemically and structurally well-characterized multidrug efflux proteins (28), and TrkH is a potassium ion transporter (29). These exact mutations have no precedent in the literature to our knowledge. Because they are missense mutations or in-frame deletions, it is unclear whether they cause loss of function or altered function (table S1). Because permissive media contain four artificial targets of efflux (BipA, l-arabinose, chloramphenicol, and SDS), mutations that confer a selective advantage during continuous evolution could disable BipA/l-arabinose efflux, improve chloramphenicol/SDS efflux, or affect transport of these or other species more indirectly. Given the strong selective pressure enforced by decreasing BipA concentration, we hypothesize that mutations observed are more likely to affect BipA transport. We also observed mutations in all evolved populations to the 23S ribosomal RNA (rRNA) gene rrlA (table S2). 23S rRNA mutations have been found to enhance tolerance for D-amino acids (30) and -amino acids (31). However, 23S rRNA mutations could also be related to increased tolerance of chloramphenicol (32).

(A) List of alleles identified through next-generation sequencing. Sequencing results originally obtained during the project identified this EmrD allele as a 33-bp deletion, which was then reconstructed in the experiment shown in (B). However, resequencing performed at the end of the project identified the allele as a 39-bp deletion and was confirmed by Sanger sequencing. A repetitive GGCGCG nucleotide sequence corresponding to G323-A324 and G336-A337 creates ambiguity about the precise positional numbering of the deletion. However, the three possible 13amino acid deletions (323335, 324336, and 325337) result in the same final protein sequence. (B) Effect of reconstructed allele in DEP progenitor on doubling time as a function of BipA concentration, normalized to the doubling time of DEP at 100 M BipA. Error bars represent the SD across technical triplicates within the same experiment.

To learn how identified transporter alleles may contribute to increased growth rates at low BipA concentration, we performed allelic reconstruction in the progenitor DEP strain using MAGE (33). Among four mutants that we generated in DEP, we observed growth of all mutants at 2 M BipA, a condition in which progenitor DEP could not grow (Fig. 3B and fig. S8). Furthermore, only emrD mutants exhibited near-normal growth at 1 M BipA. To investigate possible differential sensitivity of strains that contain reconstructed alleles to other media components of interest (SDS, l-arabinose, tris buffer, and chloramphenicol), we varied the concentration of these components and measured doubling times (fig. S9). We observed no significant deviation in doubling time from DEP in any of these cases. These results collectively suggest that observed transporter alleles are linked to BipA utilization.

The unobservable escape of DEP even after 100 days of evolution encouraged us to explore the possibility of an improved in vitro model for host-microbe interactions. In vitro models allow direct visualization and measurement of cells and effectors during processes such as pathogenesis (34). They are more relevant than animal studies for several human cell-specific interactions due to biological differences across animal types (35, 36). A nonpathogenic E. coli strain engineered to express heterologous proteins could be particularly useful for studying or identifying virulence factors and disease progression. However, an obstacle associated with coculture of microbial and mammalian cells is microbial takeover of the population. Approaches used to address this are bacteriostatic antibiotics (37), semipermeable Transwell membranes (3840), microcarrier beads (41), microfluidic cell trapping (42), peristaltic microfluidic flow (43, 44), and microfluidic perfusion (45). However, the use of a well-characterized synthetic auxotroph capable of limited persistence could offer a superior alternative for spatiotemporal control of microbial growth, especially for studying longer duration phenomena such as chronic infection or wound healing. Our study demonstrates how temporal control can be achieved by removal of BipA; we anticipate that spatial control could be achieved by patterning BipA onto a variety of solid surfaces with limited diffusion, such as a skin patch.

We investigated mammalian cell culture health, growth, and morphology after simple transient exposure to a hypermutator variant of DEP that we engineered by inactivating mutS during allelic reconstruction (DEP*). The use of DEP* rather than DEP is yet another form of a stress test to increase opportunity for escape under coculture conditions. We directly cocultured adherent human cell line human embryonic kidney (HEK) 293T with either no bacteria, nonauxotrophic E. coli DH5, or DEP* overnight (24 hours). HEK293T cells were cultured in selection media that allow only growth of desired but not contaminant strains while selecting for bacterial plasmid maintenance. After coculture, we washed cells and replenished cells with media varying in inclusion of BipA and/or an antibiotic cocktail (penicillin/streptomycin/amphotericin B). We continued incubation and imaged cells at days 2, 4, and 7 after initial coincubation. HEK293T cells contain a copy of mCherry integrated into the AAVS1 locus, and they appear red. DH5 and DEP* were transformed with Clover green fluorescent protein before coculture and appear green.

Compared to the control culture where bacteria were not added (Fig. 4A), HEK293T cells cocultured with DH5 display visible bacterial lawns with no attached human cells in the absence of the antibiotic cocktail at all days of observation (Fig. 4B). In the presence of antibiotic, cocultures containing DH5 sharply transition from bacterial overgrowth to apparent bacterial elimination (Fig. 4C). In contrast, cells cocultured with DEP* in the absence of BipA exhibited similar morphology to the control at all days of observation and no detectable bacteria by fluorescence microscopy on day 7, without the need for antibiotics to achieve bacterial clearance (Fig. 4D). Thus, DEP* addition was not detrimental to HEK293T cells in the absence of BipA, and DEP* remains biocontained and cannot survive because of cross-feeding. Clearance of bacterial cells from human cells appears to occur faster for DEP* when not provided BipA (Fig. 4D) than for DH5 when provided with the antibiotic cocktail (Fig. 4C).

Bacteria were added to HEK293T cell cultures and coincubated for 24 hours before washing and replenishing media. HEK293T cells express mCherry, whereas bacterial cells express Clover green protein marker. Images were taken at days 2, 4, and 7 after coincubation. (A) Untreated HEK293T cells. (B) HEK293T with commercial E. coli DH5 in the absence of antibiotic cocktail. (C) HEK293T with DH5 in presence of antibiotic cocktail. (D) HEK293T and DEP* (mismatch repair inactivated to create hypermutator phenotype) in the absence of BipA. (E) HEK293T cells and DEP* in the presence of BipA. (F) HEK293T and DEP* in the absence of BipA until day 2 [identical at this point to condition in (D)], and then 100 M BipA was added to this condition daily until day 7.

To learn how the synthetic auxotroph behaves when supplied its essential nutrient in these coculture settings, we tested DEP* cocultures with continual resupply of 100 M BipA. Here, DEP* proliferates and in turn decreases proliferation and viability of HEK293T cells (Fig. 4E). A bacterial lawn begins to form on day 2, and at later times, human cell debris is overtaken by DEP*. This demonstrates that DEP* is fully capable of taking over the coculture if supplied with BipA. Replicates for these experiments can be found in figs. S10 to S12.

Given that DEP* grows in cocultures when BipA is provided, we sought to understand whether it could be rescued by readdition of BipA after multiple days of withholding. The possible time scale of reemergence influences applications where the duration of bacterial activity would need to be prolonged and/or repeated via limited BipA introduction while remaining contained. We find that coculturing DEP* with HEK293T cells for 2 days in the absence of BipA followed by the addition of BipA at day 2 does not rescue the DEP* growth (Fig. 4F and fig. S13). Human cells still grow and look morphologically similar to untreated cells, and bacteria are not visible. To look at analogous questions for nonauxotrophic E. coli, we removed antibiotics after 2 days of coculturing and do not observe bacterial rescue (fig. S13). We also investigated whether bacterial clearance could be delayed by the addition of antibiotic after some growth of DH5. DH5 cells grown in the absence of the antibiotic cocktail for 2 days before addition of the cocktail and maintenance to day 7 result in bacterial lawns (fig. S13, A and D). This demonstrates that antibiotic cocktails ordinarily used in mammalian cell culture maintenance can become ineffective beyond a certain amount of nonauxotrophic bacterial growth, whereas synthetic auxotrophy is subject to fewer and different constraints.

To further investigate the persistence of progenitor DEP and its evolved descendants, we performed BipA readdition studies in Lennox lysogeny broth (LB-Lennox) monoculture. Within 7 hours of BipA removal, DEP cell populations that are harvested from midexponential or stationary phases can be reactivated upon delayed BipA addition with unperturbed growth kinetics after a highly tunable lag phase (fig. S14). Further studies are ongoing to investigate the amount of time after which BipA reintroduction can recover growth of synthetic auxotrophs under different contexts.

We have shown that synthetic auxotrophy can exhibit long-term stability and function in unique contexts, enabling reliable control of microbial proliferation. Recent work has also shown that the escape rate and fitness of multiple synthetic auxotrophs can be improved by increasing the specificity of nsAA incorporation machinery (46). Collectively, these engineering and characterization efforts advance synthetic auxotrophy as a powerful safeguard for basic and applied research when using engineered microbes.

Cultures for general culturing, growth rate assays, biocontainment escape assays, MAGE, and fluorescent protein assays were prepared in LB-Lennox medium [bacto tryptone (10 g/liter), sodium chloride (5 g/liter), and yeast extract (5 g/liter)] supplemented with chloramphenicol (15 g/ml), 0.2% (w/v) l-arabinose, 20 mM tris-HCl buffer, 0.005% SDS, and variable concentration of L-4,4-biphenylalanine (BipA). Unless otherwise indicated, all cultures were grown in 96-well deep plates in 300 l of culture volumes at 34C and 400 rpm. The above media are permissive for growth of the synthetic auxotroph. Nonpermissive media are identically formulated as permissive media except for BipA, which is not included.

Construction of appropriate fluidics and chambers followed the eVOLVER framework (26) (figs. S1 and S2). The following components were included: (i) fluidics and chambers (reactor vial, inlet and outlet lines, filters, pumps, stirrers, and inlet and outlet reservoirs); (ii) light source and detector (LED and photodiode); (iii) controller hardware (circuit and microprocessors); and (iv) controller software (Arduino for controlling tasks, Raspberry Pi for computing tasks, and Python code for programming tasks) (full build of materials included in table S3). Briefly, our apparatus consisted of a custom smart sleeve (fig. S3), with the following modifications: Each vial was constructed without temperature control and was supplied by two media pumps (one for permissive media and another for nonpermissive media) and connected to one waste pump. All pumps were RP-Q1 from Takasago Fluidics, each driven off a standard N power MOSFET (metal oxide semiconductor field-effect transistor) with an Arduino controlling the gate. Like the eVOLVER system, we installed a stirring fan underneath each sleeve that consisted of magnets attached to a computer fan. By including a small stir bar within each reactor vial, we enabled efficient mixing of 1-ml working volumes. To enable automated measurement of turbidity [optical density (OD)], we used a 605-nm LED (LO Q976-PS-25) and an OPT101P-J photodiode detector. We mounted the LED and detector on custom printed circuit boards mounted to the vial sleeve to enable easier construction and better control of ambient light leakage into the light path (fig. S4). To monitor turbidity within each vial and to control pump arrays in response, we constructed printed circuit board designs in Gerber format as is standard for circuit fabrication. We attached an Arduino Mega microcontroller with an analog-digital converter and directed it using a PyMata script (47).

Chemostats were operated by automated maintenance of culture OD within a specified parameter range within exponential growth phase (20 to 80% of dynamic range) depending on linearity of photodiode measurements. Constant fixed dilutions of permissive media were used to decrease OD until desired equilibrium of cell growth and dilution rates. This resulted in a sawtooth curve (27), where time between peaks is recorded as a proxy for growth rate. Our program gradually decreased the ratio of permissive to nonpermissive media as step functions, with a specified number of dilution cycles allowed to elapse before the next decrease to provide time for acclimation. Time between OD peaks lengthened as strain fitness decreased. Once a threshold difference between ancestral peak-to-peak time and current peak-to-peak time was passed, the ratio of permissive to nonpermissive media remained fixed. This allowed cells to evolve until peak-to-peak time returns to ancestral values, which initiated the next phase of decrease in BipA concentration. To assess the quality of our continuous evolution process, we paused chemostat trials on a weekly basis for strain storage, strain evaluation, chemostat cleaning, and investigation of contamination.

Growth assays were performed by plate reader with blanking as previously described (25). Overnight cultures were supplemented with different BipA concentrations depending on the strain. The DEP progenitor strain was grown in permissive media containing 100 M BipA, and evolved DEP strains DEP.e3, DEP.e4, and DEP.e5 were grown in permissive media containing 1 M BipA. Saturated overnight cultures were washed twice in LB and resuspended in LB. Resuspended cultures were diluted 100-fold into three 150-l volumes of permissive media. BipA concentrations used in this assay were 0, 0.001, 0.01, 0.1, 0.5, 1, 10, and 100 M. Cultures were incubated in a flat-bottom 96-well plate (34C, 300 rpm). Kinetic growth (OD600) was monitored in a Biotek Eon H1 microplate spectrophotometer reader at 5-min intervals for 48 hours. The doubling times across technical replicates were calculated as previously indicated. We refer to these as technical replicates because although triplicate overnight cultures were used to seed triplicate experiment cultures, the overnight cultures were most often seeded from one glycerol stock.

Escape assays were performed as previously described with minor adjustments to decrease the lower detection limit for final evolved populations (23, 46). Strains were grown in permissive media and harvested in late exponential phase. Cells were washed twice with LB and resuspended in LB. Viable CFU were calculated from the mean and SEM of three technical replicates of 10-fold serial dilutions on permissive media. Twelve technical replicates were plated on noble agar combined with nonpermissive media in 500-cm2 BioAssay Dishes (Thermo Fisher Scientific 240835) and monitored daily for 4 days. If synthetic auxotrophs exhibited escape frequencies above the detection limit (lawns) on nonpermissive media, escape frequencies were calculated from additional platings at lower density. The SEM across technical replicates of the cumulative escape frequency was calculated as previously indicated.

Genomic DNA was obtained from evolved populations and ancestral clone using the Wizard Genomic DNA purification kit (Promega). Sequencing libraries were prepared as described in Baym et al. (48). Sequencing was performed using a NextSeq instrument, producing 75base pair (bp), paired-end reads. Resulting data were aligned to the E. coli C321.delA nonauxotrophic but recoded reference sequence (GenBank no. CP006698.1) and the sequence of the plasmid encoding nsAA incorporation machinery. The Millstone software suite was used to identify variants, provide measures of sequencing confidence, and predict their likelihood of altering gene function (49). Genomic variants of low confidence, low sequence coverage, or presence in the ancestral strain were discarded, prioritizing variants observed in three nonessential genes that encode membrane proteins: acrB, emrD, and trkH.

Subsequent genomic sequencing was performed on genomic DNA extracted from the evolved populations and ancestral clone using the DNeasy Blood and Tissue Kit (Qiagen). Genomic DNA was then sent to the Microbial Genome Sequencing Center (MiGS) in Pittsburgh, PA. Variants were identified through the variant calling service from MiGS.

MAGE (33) was used to inactivate the endogenous mutS gene in the DEP strain. Overnight cultures were diluted 100-fold into 3 ml of LB containing chloramphenicol, BipA, l-arabinose, and tris-HCl buffer and grown at 34C until midlog. The genome-integrated lambda Red cassette in this C321.A-derived strain was induced in a shaking water bath (42C, 300 rpm, 15 min), followed by cooling the culture tube on ice for at least 2 min. The cells were made electrocompetent at 4C by pelleting 1 ml of culture (8000 rcf, 30 s) and washing thrice with 1 ml of ice-cold 10% glycerol. Electrocompetent pellets were resuspended in 50 l of dH2O containing the desired DNA; for MAGE oligonucleotides, 5 M of each oligonucleotide was used. Allele-specific colony polymerase chain reaction (PCR) was used to identify desired colonies resulting from MAGE as previously described (50). Oligonucleotides used for MAGE and for allele-specific colony PCR are included in table S4.

This assay was performed using a similar protocol as described in the Measurement of doubling times section. The cultures for DEP and its single mutants were grown overnight in 100 M BipA. Then, cultures were diluted 100 in the media specified. Those conditions include standard media conditions and single component changes: 0% SDS, 0.01% SDS, 0.02% (w/v) arabinose, 0 mM tris-HCl, and chloramphenicol (30 g/ml). The cultures were grown in triplicate for each condition and in a SpectraMax i3 plate reader, shaking at 34C for 24 hours. The OD600 was measured about every 5 min. The doubling times were then calculated as previously described.

HEK293T cells containing one copy of mCherry marker (red) integrated into the AAVS1 locus were grown at 40 to 50% confluency in DMEM (Dulbeccos modified Eagles medium) high-glucose medium (Thermo Fisher Scientific, catalog no. 11965175) with 10% inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, catalog no. 10082147), 100 MEM NEAA (nonessential amino acids; Thermo Fisher Scientific, catalog no. 11140050), and 100 diluted anti-anti cocktail [antibiotic-antimycotic: penicillin (10,000 U/ml), streptomycin (10,000 g/ml), and Gibco amphotericin B (25 g/ml); Thermo Fisher Scientific, catalog no. 15240112). Commercially acquired E. coli DH5 bacteria were used as control to the E. coli DEP mutS or DEP* strain. A plasmid containing Clover (green marker) containing a UAA stop codon compatible with the biocontained strain DEP, and under the selection marker ampicillin was transformed into both DH5 and DEP* strains to visualize them with the mammalian cells (red). BipA-dependent auxotroph DEP* bacteria were grown to an OD of 0.6 in LB medium supplemented with 1% l-arabinose, 100 M BipA, carbenicillin (100 g/ml), and chloramphenicol (25 g/ml) and then washed three times with 1 phosphate-buffered saline (PBS). DEP* culture conditions with l-arabinose, carbenicillin, and chloramphenicol supplements did slightly affect HEK293T early cell growth compared to untreated cells, although insufficient to affect conclusions drawn from these experiments. DH5 strain was grown to an OD of 0.6 with carbenicillin (100 g/ml). The pellet of 10-ml bacterial cell culture was resuspended in mammalian cell medium as described above without any antibiotics and anti-anti, and split equally among all conditions and their replicates. Auxotroph bacteria are added to HEK293T cells plated in pretreated 12-well plates in 2 ml of mammalian cell medium. The coculture is incubated overnight before the medium that contains the bacterial cells is removed. HEK293T cells were washed three times with 1x PBS (Thermo Fisher Scientific, catalog no. 10010023) and replenished with fresh media as conditions indicate. Media were replaced and added fresh to all conditions daily for 7 days. Imaging of cells was done with the inverted microscope Nikon Eclipse TS100 at days 2, 4, and 7 after initial coculture at 200 magnification.


Control: HEK293T grown in regular 10% FBS media with anti-anti and NEAA as described above.

DH5: HEK293T cells cocultured with this strain in mammalian cell media supplemented with carbenicillin (100 g/ml) to maintain plasmid during growth and absence of anti-anti.

DH5; anti-anti (antibiotic cocktail): HEK293T cells cocultured with this strain in mammalian cell media supplemented with carbenicillin (100 g/ml) to maintain plasmid during growth and presence of anti-anti cocktail.

DH5; anti-anti after day 2: HEK293T cells cocultured with this strain in mammalian cell media supplemented with carbenicillin (100 g/ml) to maintain plasmid during growth and absence of anti-anti cocktail. At 48 hours, anti-anti added and maintained to day 7.

DH5; anti-anti; no anti-anti after day 2: HEK293T cells cocultured with this strain in mammalian cell media supplemented with carbenicillin (100 g/ml) to maintain plasmid during growth and presence of anti-anti until day 2. After day 2, no anti-anti added and maintained to day 7.

DEP*: HEK293T cells cocultured with the biocontained strain in media supplemented with l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. No bipA or anti-anti was added.

DEP*; bipA: HEK293T cells cocultured with the biocontained strain in media supplemented with l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. One hundred micromolar bipA and no anti-anti added.

DEP*; bipA after day 2: HEK293T cells cocultured with the biocontained strain in media supplemented with l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. No bipA or anti-anti added. At 48 hours, bipA at 100 M concentration added and maintained to day 7.

DEP*; anti-anti: HEK293T cells cocultured with the biocontained strain in media supplemented with anti-anti, l-arabinose, chloramphenicol (25 g/ml), and carbenicillin (100 g/ml) to maintain bacteria and green marker. No bipA added.


Persistence was evaluated by two kinds of assays: plate reader and colony count. For the plate reader case, DEP, DEP.e3, DEP.e4, and DEP.e5 cultures were grown overnight in permissible media conditions with 100 M BipA. For cells harvested at midexponential phase, the cultures were diluted 100 and grown to that state. Both stationary-phase and midexponential-phase cultures were then washed twice with LB media and resuspended in the original volume of nonpermissible media containing all specified media components except BipA. The resuspended cultures were then diluted 100 into nonpermissible media in triplicate for each time point to be tested. The specified concentration of BipA was then added back to those cultures at the specified time points. Typically, the BipA readdition occurred at 10 or 5 M concentrations and at hourly or daily intervals. The cultures were then incubated with shaking in SpectraMax i3 plate readers in a flat, clear-bottom 96-well plate with breathable and optically transparent seal for an upward of 84 hours at 34C. Approximately every 5 min, the OD600 was measured to determine cell growth kinetics.

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells - Science Advances

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