Growing Unknown Microbes One by One

A new technique developed at Caltech helps grow individual species of the unknown microbes that live in the human body.

Trillions of bacteria live in and on the human body; a few species can make us sick, but many others keep us healthy by boosting digestion and preventing inflammation. Although there's plenty of evidence that these microbes play a collective role in human health, we still know very little about most of the individual bacterial species that make up these communities. Employing the use of a specially designed glass chip with tiny compartments, Caltech researchers now provide a way to target and grow specific microbes from the human gut—a key step in understanding which bacteria are helpful to human health and which are harmful.

The work was published the week of June 23 in the Proceedings of the National Academy of Sciences.

Although a few bacterial species are easy to grow in the laboratory, needing only a warm environment and plenty of food to multiply, most species that grow in and on the human body have never been successfully grown in lab conditions. It's difficult to recreate the complexity of the microbiome—the entire human microbial community—in one small plate (a lidded dish with nutrients used to grow microbes), says Rustem Ismagilov, Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering at Caltech.

There are thousands of species of microbes in one sample from the human gut, Ismagilov says, "but when you grow them all together in the lab, the faster-growing bacteria will take over the plate and the slow-growing ones don't have a chance—leading to very little diversity in the grown sample." Finding slow-growing microbes of interest is like finding a needle in a haystack, he says, but his group wanted to work out a way to "just grow the needle without growing the hay."

To do this, Liang Ma, a postdoctoral scholar in Ismagilov's lab, developed a way to isolate and cultivate individual bacterial species of interest. He and his colleagues began by looking for bacterial species that contained a set of specific genetic sequences. The targeted gene sequences belong to organisms on the list of "Most Wanted" microbes—a list developed by the National Institutes of Health (NIH) Human Microbiome Project. The microbes carrying these genetic sequences are found abundantly in and on the human body, but have been difficult to grow in the lab.

To grow these elusive microbes, the Caltech researchers turned to SlipChip, a microfluidic device previously developed in Ismagilov's lab. SlipChip is made up of two glass slides, each the size of a credit card, that have tiny etched grooves which become channels when the grooved surfaces are stacked atop one another. When a sample—say, a jumbled-up assortment of bacteria species collected from a colonoscopy biopsy—is added to the interconnected channels of the SlipChip, a single "slip" of the top chip will turn the channels into individual wells, with each well ideally holding a single microbe. Once sequestered in an isolated well, each individual bacterium can divide and grow without having to compete for resources with other types of faster-growing microbes.

The researchers then needed to determine which compartment of the SlipChip contained a colony of the target bacterium—which is not a simple task, says Ismagilov. "It's a Catch-22—you have to kill the organism in order to find its DNA sequence and figure out what it is, but you want a live organism at the end of the day, so that you can grow and study this new microbe," he says. "Liang solves this in a really clever way; he grows a compartment full of his target microbe in the SlipChip, then he splits the compartment in half. One half contains the live organism and the other half is sacrificed for its DNA to confirm that the sequence is that of the target microbe."

The method of creating two halves in each well in the SlipChip will be published separately in an upcoming issue of the journal Integrative Biology.

To validate the new methodology, the researchers isolated one specific bacterium from the Human Microbiome Project's "Most Wanted" list. The investigators used the SlipChip to grow this bacterium in a tiny volume of the washing fluid that was used to collect the gut bacteria sample from a volunteer. Since bacteria often depend on nutrients and signals from the extracellular environment to support growth, the substances from this fluid were used to recreate this environment within the tiny SlipChip compartment—a key to successfully growing the difficult organism in the lab.

After growing a pure culture of the previously unidentified bacterium, Ismagilov and his colleagues obtained enough genetic material to sequence a high-quality draft genome of the organism. Although a genomic sequence of the new organism is a useful tool, further studies are needed to learn how this species of microbe is involved in human health, Ismagilov says.

In the future, the new SlipChip technique may be used to isolate additional previously uncultured microbes, allowing researchers to focus their efforts on important targets, such as those that may be relevant to energy applications and the production of probiotics. The technique, says Ismagilov, allows researchers to target specific microbes in a way that was not previously possible.

The paper is titled "Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa." In addition to Liang and Ismagilov, other coauthors include, from Caltech, associate scientist Mikhail A. Karymov, graduate student Jungwoo Kim, and postdoctoral scholar Roland Hatzenpichler, and, from the University of Chicago department of medicine, Nathanial Hubert, Ira M. Hanan, and Eugene B. Chang. The work was funded by NIH's National Human Genome Research Institute. Microfluidic technologies developed by Ismagilov's group have been licensed to Emerald BioStructures, RanDance Technologies, and SlipChip Corporation, of which Ismagilov is a cofounder.

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40-Year Service Awardees

Caltech Staff Service Awards 2014

The 59th Annual Staff Service Awards will be presented in Beckman Auditorium on Monday, June 2, at 10 a.m. During the ceremony, more than 250 staff members whose service ranges from 10 to 50 years will be honored. A full list of awardees can be found here.

This week we are featuring Caltech staff members who will be recognized for 40 and 45 years of service to the Institute.


The honorees include three 40-year staff members: Eugene Akutagawa, a senior scientist in biology and a member of the professional staff; Susi Martin, assistant to the Board of Trustees; and Steve Vass, a senior instrument specialist at the Laser Interferometer Gravitational-wave Observatory (LIGO).


Eugene Akutagawa graduated from UCLA with a bachelor's degree in microbiology; a help-wanted ad in the Los Angeles Times for a lab assistant brought him to Caltech, where "I was standing in the hallway, waiting to be interviewed, and there's [Nobel Laureate] Max Delbrück coming out of the lab. To me, a microbiologist, he was like a god, and there he was, right in my face—so I knew this place was going to be great. And I liked its smallness, especially contrasted with UCLA, where undergraduate biology classes were 700 or 800 people spilling into the aisles."

Nevertheless, his first job proved unrewarding. "I was implanting electrodes in rats and watching them press the lever until they pooped out," Akutagawa recalls. (The lab belonged to Research Associate Marianne Olds, whose husband, Bing Professor of Behavioral Biology James Olds, had discovered the brain's pleasure center more than two decades earlier.) "Then one day, I was sitting in the parking lot eating my lunch, and a mockingbird landed on a bush and started singing his heart out. I thought, 'I know he's not really singing words, but he's communicating. It would be interesting to study that.' And lo and behold, within a few months Mark Konishi [now the Bing Professor of Behavioral Biology, Emeritus] came here from Princeton."

Konishi had already made a name for himself studying songbirds and owls, so Akutagawa changed labs. The job interview was informal, Akutagawa recalls. "Mark said, 'What experience do you have?' And I said, 'Well, when I was growing up in Hawaii, I tried to save nestlings that had fallen out of their nests.' And he looked at me very sternly and said, 'What did you feed them?' I said, 'Rice. And water.' 'Did any of them live?' 'Nope. They all died.' I think he appreciated my honesty. He never told me I got the job, but he went over to the chalkboard and drew a football shape. He said, 'That's canary seed. That goes to canaries and white-crowned sparrows.' And he drew a little circle, and he says, 'That's millet. That goes to the finches.'"

Within a decade Akutagawa had become a full-fledged collaborator, doing the meticulous microscopy needed to trace fine neural circuitry. In 1985, Konishi and Akutagawa published a paper that showed why male zebra finches sing and females don't: specialized neurons in the male's brain flourish and develop many connections, but in females they atrophy and die. Says Akutagawa, "Our relationship eventually evolved into me doing my own independent research. It's been quite a ride, I must say." The ride, however, is nearing its end; Konishi has retired, and Akutagawa will be following suit.

"I love this job," Akutagawa continues. "It's more like a hobby. It's just an amazing place to work, in large part because Mark was just an incredible supervisor. He gave us a lot of freedom, which spurs a lot of good science."


Susi Martin works in the Caltech president's office as assistant to the Board of Trustees. The Board has 85 members and meets five times a year, and Martin manages their comings and goings. She says, "I arrange transportation to and from the airport, hotels, whatever assistance they need. It could be anything." During her tenure, she's moved from a Selectric typewriter to a Filemaker Pro database to the Internet; from three-ring binders to PDFs.

Martin began her Caltech career in the procurement division at JPL before joining the office of then-director Bruce Murray. After a special assignment supporting the Seasat mission's Failure Review Board in 1978, she moved on to one of JPL's early biomedical technology projects before transferring to the Lab's office of planning and review. It was there in April, 1981 that Hardy Martel (BS '49, PhD '56), an electrical engineering professor and the secretary to Caltech's Board of Trustees, called to inquire whether she'd consider moving to campus. One of Martin's former colleagues in the director's office, Mary Webster, had joined the staff of Caltech president Marvin Goldberger earlier that year; when Martel became in need of an assistant, Webster had recommended Martin.

From 1988 to 1994, Martin also served first as assistant secretary and then secretary to the board of directors of the California Association for Research in Astronomy (CARA), a partnership set up by Caltech and the University of California to build and operate the W. M. Keck Observatory on the summit of Mauna Kea.

"I love working with the trustees," Martin says. "It is an honor and a delight—they are a truly remarkable group of individuals, and it is a privilege to facilitate their work on behalf of Caltech." Over the past 33 years, no two days have been the same, she says. "The challenges have been interesting, but the rewards have been awesome and tremendously diverse." For example, Martin was at JPL for the landing of the Mars rover Curiosity, staffing one of the rooms set up for the trustees. "Seeing that first image of the rover's shadow cast on the surface was just amazing," she recalls. "To see something nobody else had ever seen—to be a part of that history—was so cool."


While Akutagawa and Martin have essentially stayed put, Steve Vass has occupied eight different offices in three of Caltech's academic divisions. Vass was born and raised in Hungary, where he learned electronics at a trade school. "I had some college but not too much." He eventually came to the United States, where he landed a job in Caltech's biology division in the laboratory of then-professor Leroy Hood (BS '60, PhD '68). Vass helped Hood and postdoc Michael Hunkapiller (PhD '74) build the protein sequenator, which automatically determines the sequence of amino acids that make up a protein. Two decades later, this machine and the other ones developed in the Hood lab—the protein synthesizer, the DNA synthesizer, and the DNA sequenator—would spark the biotech revolution of the 1990s.

In the early 1980s, Vass moved to the Division of Chemistry and Chemical Engineering, where he built X-ray diffractometers for physical chemist Richard Dickerson. Dickerson used them to make high-precision measurements of DNA's crystal structure—both its usual right-handed spiral and the less common left-handed form.

In 1987, Vass moved again—to the Division of Physics, Mathematics and Astronomy and the LIGO project. LIGO searches for the gravitational waves that Einstein predicted would be generated by the motions of extremely massive bodies—colliding black holes being an oft-cited example. The detector consists of twin interferometers, each with a perpendicular set of 4-kilometer-long arms, that were built in Louisiana and Washington in the late 1990s. When Vass joined the project, the design's details were being worked out in a prototype interferometer with 40-meter arms that had been built on the Caltech campus. Nearly two decades later, the 40-meter prototype remains the proving ground for next-generation ideas.

Vass describes how LIGO changed his perspective: "In biology, people said, 'Oh, if we only had a good chemist, we would hit it out of the park.' Then in chemistry they said, 'Oh, if we had a really good electronics guy, we would be just the best.' But in physics, they say, 'We know everything. We can do it ourselves.'"

"Basically, I run the lab, but the fun part is you get to do everything. This morning, I've been hunting for 'ground loops.' The east end of the interferometer has a 60-Hertz hum, which is line current, and it's ruining the spectrum. So I'm going around with an ohmmeter looking for something disconnected—or something connected that shouldn't be. My job is to prepare the best possible environment to get good science done."

LIGO measures the distance between suspended mirrors to within a billionth of the diameter of an atom by bouncing a laser beam between them, so Vass begins his mornings making sure the interferometer hasn't lost lock. "If people stayed really late the night before, things will be fine," he says. "But if they left at 10 p.m., everything will have drifted a little. And earthquakes affect the machine. It's much better designed against quakes now, but in earlier days if we had a local magnitude 4, our precious glass might have fallen and gotten chipped, or our mirror coating could have been ruined. Back then it was a baby, and I've seen it grow up with my kids. I have grandkids now, and someday LIGO will produce something, too—some cosmic event will happen close by, and we'll see it."

"I have to say thank you to all the people who've helped me grow," Vass concludes. "I've learned a lot here and had a lot of fun doing it."

Douglas Smith
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Ditch Day? It’s Today, Frosh!

Today we celebrate Ditch Day, one of Caltech's oldest traditions. During this annual spring rite—the timing of which is kept secret until the last minute—seniors ditch their classes and vanish from campus. Before they go, however, they leave behind complex, carefully planned out puzzles and challenges—known as "stacks"—designed to occupy the underclass students and prevent them from wreaking havoc on the seniors' unoccupied rooms.

Follow the action on Caltech's Facebook and Twitter pages as the undergraduates tackle the puzzles left around campus for them to solve, and get in on the conversation by sharing your favorite Ditch Day memories. Be sure to use #CaltechDitchDay in your tweets and postings.

View photos from the day:


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