Programming an Abundant Human Gut Microbe

By Aaron Krol 

July 24, 2015 | If you grew up playing with plug-and-play electronics kits (or, these days, the magical redstone in Minecraft), you’ll be familiar with the brunt literal-mindedness of circuit boards. Whatever elaborate construct of LEDs, buzzers, and motors you come up with has to rely on just two input conditions: is there electricity, or no?

No one ever accused the genome of being that simple, but certain genetic elements do behave much like the circuits on a circuit board. In the genomes of many organisms, “inducible promoters” have evolved to be single-mindedly concerned with one aspect of the environment: is there light around? Is the temperature above a certain threshold? Have I eaten a particular nutrient? If the answer is yes, these promoters drastically ramp up the activity of nearby genes they’re attached to, creating proteins that help the organism respond to its changing environment.

To a certain breed of biologist, finding that kind of on-off switch in the genome is an invitation to tinker.

At the MIT Synthetic Biology Center, Timothy Lu is a specialist in “genetic circuits,” reengineering bacteria to add new functions that can be controlled with environmental cues. Building on in-depth knowledge of the genomes of model organisms like E. coli, whose DNA architecture has been extensively studied for decades, Lu and his colleagues have cut and pasted inducible promoters to attach them to new genes ― like moving a battery on a circuit board to hook it to a new servo or lightbulb. When given the right signals, these engineered E. coli can perform tasks like creating random stretches of DNA as a primitive memory system, or spinning useful synthetic fibers.

Now, the Synthetic Biology Center has built similar circuits in a new, and in many ways more significant, organism, Bacteroides thetaiotaomicron (B. theta for short). The work was published earlier this month in Cell Systems, with Lu as senior author joined by Mark Mimee, Alex Tucker, and Christopher Voigt. Lu believes his team’s redesigned B. theta brings the field of synthetic biology tangibly closer to making organisms that can perform useful services inside the human body.

Unlike E. coli, B. theta is not a mainstay of laboratory science. While its genome has been fully known since 2003, and it grows readily enough in the lab, its study is mostly reserved for specialists in the microbiota of the human gut, where it lives. In its natural environment, however, B. theta dwarfs E. coli, both in numbers and in importance to the ecosystem. Exquisitely adapted to breaking down complex sugars in the colon, B. theta is one of our most abundant fellow travelers: a single gram of our feces may contain several billion of them.

If scientists want to program bacteria to live and work in the human gut, says Lu, species like B. theta will be the best ambassadors. “They are present at high levels and can stably colonize a wide range of different people,” he tells Bio-IT World. “Imagine having engineered bacteria living in your gut that can give you an early warning of colon cancer or an inflammatory bowel disease flare. We believe that this platform will enable new non-invasive diagnostics and therapeutics for human disease.”

Not everyone agrees that engineering B. theta is such a large leap from past accomplishments. Pamela Silver, a prominent synthetic biologist at Harvard Medical School, argues that this organism is actually fairly similar to E. coli in that many of its genetic functions have already been mapped out. “Because of its prior characterization, B. theta was low hanging fruit,” she says.

Nonetheless, Silver, who was not involved in the Cell Systems paper, is pleased to see a new and naturally abundant species fitted with genetic circuits. “I suspect that every gut species will offer a unique opportunity,” she says. “We are just at the start of an exciting time, and the parts generated here will be part of that story.”

Under the Hood 

As is often the case in the life sciences, the road to ambitious new therapies runs through lab mice.

Not all genetic circuits work inside animals. Not only do your reprogrammed organisms need to survive in the complex environment of the gut, but the signals you use to turn their circuits on and off have to reach them. It’s hard to shine a light in a mouse’s stomach.

Chemical signals, however, work well, as long as you choose molecules that can safely be fed to mice. “Prior efforts showed that it is possible to engineer model organisms that can function in the gut of animals,” says Lu. To repeat that feat with B. theta, Lu’s team focused on chemically-induced promoters.

Synthetic biologists have already compiled a large library of these promoters, but they need tweaking before they can be inserted into a new organism. In the lingo of synthetic biology, a bacterium like E. coli is a “chassis,” like the body of a car or cell phone ― but that’s a poor metaphor for something as complex as a living species. Software doesn’t care if it’s operating in a Samsung or a Motorola, but genetic circuits have to interact with thousands of other elements in their host genomes.

“In electronics, one can take discrete parts like resistors or transistors, put them on a substrate where they are all isolated from each other, and then hook up the parts,” says Lu. “In biology, many of the synthetic parts we use are molecules that are constantly bumping into each other inside of the cell, and thus making specific circuits or hooking up specific parts with each other is harder.”

The MIT team took several approaches to making this complex web of circuits work in a new chassis. In some cases, they chose new inducible promoters from B. theta’s native genome, including one that responds to a kind of sugar called rhamnose. That promoter was duplicated and coupled with a newly inserted gene to add a new function.

In other cases, the researchers copied promoters from E. coli, modifying the DNA slightly to look more like a B. theta promoter so the new host’s cellular machinery could recognize and use the added parts. The team also made use of a big recent development in genetic engineering, a molecular system called CRISPR interference. CRISPR systems involve enzymes that can hunt down specific sequences of DNA and latch onto them ― in the case of CRISPR interference, staying in place to shut down their ability to make proteins.

“The major advantage of CRISPR is that once it’s established in a new host, it can be easily reprogrammed to target any gene in the organism,” says Lu. In his lab’s engineered B. theta, CRISPR interference was used as a sort of “off switch” for a separate genetic circuit, targeting a gene that makes a fluorescent molecule called luciferase. When mice carrying the engineered B. theta strain were fed a molecule called arabinogalactan, the bacteria turned on production of luciferase, which could be seen in the rodents’ fecal pellets. Adding a second molecule, IPTG, to the animal feed activated CRISPR, turning the production of luciferase off again.

The variety and flexibility of circuits that Lu and his colleagues added to B. theta shows that the barriers to synthetic biology are quickly getting lower, as we learn more about the basic pieces of bacterial genomes. “Promoter structure and regulation may differ among species, but the general rules will hold,” says Silver. “We are really good at figuring out gene regulation ― this is the foundation of molecular biology.”

Putting Bacteria to Work 

As a first proof of principle, the strain of B. theta that Lu and his colleagues engineered is not exactly a medical marvel. The new genes they added to its DNA ― like the one that generated fluorescent mouse pellets ― were chosen to be easily measured, not to give its rodent hosts remarkable new healing powers.

Nonetheless, these experiments are notable achievements, and not only because they were brought off successfully. Lu’s team put several of their circuits together in the same bacteria, making the B. theta used in their animal experiments one of the more intricate examples of genetic circuits yet demonstrated. The strain also contains an example of a “memory switch,” in which a temporary signal produces a permanent change in the genome. In this case, B. theta’s rhamnose-inducible promoter was rewired to an enzyme called integrase, which takes a separate, non-functional part of the genome and flips it over.

“In this work, we did not design a phenotype to result from the memory switches,” says Lu. “However, our prior work has shown that complex gene programs could be created with recombinase-based genetic memory. These gene programs could have any arbitrary outputs.” A potential application is a bacterium that acts as a sort of watchdog for pathogens or environmental contaminants, producing a “reporter” molecule after exposure.

Lu is eager to move on to more complex circuitry ― including the kinds of “logic gates” that make up sophisticated electronics. For instance, he imagines that a bacterium that needs to encounter multiple signals before its circuits are turned on could be a useful diagnostic.

“In many complex diseases, a single biomarker is insufficient,” he says. “We may want bacteria to sense multiple biomarkers, and only when all of them are present should it declare that the disease is manifesting.”

Such handy engineered bacteria are still a little ahead of the science. “This story is a tour de force of engineering, but with not much new learned,” says Silver, who has pulled off similar experiments with E. coli. “Useful tools have been generated that hopefully will be of general use to the community. It is also nice to see another engineered genetic circuit perform in an animal.”

But she also cautions that most of the denizens of the human intestinal tract are much more mysterious than B. theta. In many cases, they don’t grow in the lab, and their genomes have never been fully assembled. With these species, there are no pre-developed tools for manipulating the genome ― in important ways, we’re no closer than before to modifying most of the important organisms in the human microbiome.

Then again, with the latest tools for engineering the genome, it may be possible to create simple circuits without knowing too much about the regulatory quirks of each new organism’s DNA. “CRISPR seems to have widespread applicability,” Silver says, “and so may hold the key to engineering many species ― as is being demonstrated over and over.”

Synthetic biologists are getting more confident, and more ambitious. And with thousands more species of intestinal bacteria waiting to be reengineered, there are surely some interesting new circuits out there to stack on the board.