E. Coli Bacteria as Medical Sensors and Hard Drives?

E.Coli
Modified E. coli bacteria can serve as sensors and data storage devices for environmental and medical monitoring. Credit: Centers for Disease Control and Prevention. View larger image

E. coli bacteria help us digest our food, produce vitamin K and have served as a model organism in research for decades. Now, they might one day be harnessed as environmental or medical sensors and long-term data storage devices Exit icon.

MIT researchers Timothy Lu Exit icon and Fahim Farzadfard modified the DNA of E. coli cells so that the cells could be deployed to detect a signal (for example, a small molecule, a drug or the presence of light) in their surroundings. To create the modified E. coli, the scientists inserted into the bacteria a custom-designed genetic tool.

When exposed to the specified signal, the tool triggers a series of biochemical processes that work together to introduce a single mutation at a specific site in the E. coli’s DNA. This genetic change serves to record exposure to the signal, and it’s passed on to subsequent generations of bacteria, providing a continued record of exposure to the signal. In essence, the modified bacteria act like a hard drive, storing biochemical memory for long periods of time. The memory can be retrieved by sequencing the bacteria or through a number of other laboratory techniques.

The researchers call the new technology SCRIBE, for Synthetic Cellular Recorders Integrating Biological Events. It’s not only able to detect the presence of a chosen signal, but also the magnitude and duration of exposure.

SCRIBE could be used to indicate chemical contaminants, pollutants or other substances in the environment. Conceivably, it could also serve as a sensor for virtually any molecule in the body—blood proteins, dietary products, immune factors, hormones, disease markers, pharmaceuticals, toxins—to monitor health, detect disease and track the progress of countless medical conditions.

If you’re interested in SCRIBE’s technical details, which include harnessing an enzyme from another type of bacteria, producing a specific RNA-DNA hybrid and generating single-stranded DNA containing the desired mutation, you can read a perspective Exit icon on the paper or watch an animation Exit icon of the process.

This work was funded in part by NIH under grants P50GM098792 and DP2OD008435.

Field Focus: Bringing Biology Into Sharper View with New Microscopy Techniques

Composite image of mitochondria in a cell
In this composite image of mitochondria in a cell, the left panel shows a conventional optical microscopy image, the middle panel shows a three-dimensional (3-D) STORM image with color indicating depth, and the right panel shows a cross-section of the 3-D STORM image. Credit: Xiaowei Zhuang laboratory, Howard Hughes Medical Institute, Harvard University. View larger image.

Much as a photographer brings distant objects into focus with a telephoto lens, scientists can now see previously indistinct cellular components as small as a few billionths of a meter (nanometers). By overcoming some of the limitations of conventional optical microscopy, a set of techniques known as super-resolution fluorescence microscopy has changed once-blurry images of the nanoworld into well-resolved portraits of cellular architecture, with details never seen before in biology. Reflecting its importance, super-resolution microscopy was recognized with the 2014 Nobel Prize in chemistry.

Using the new techniques, scientists can observe processes in living cells across space and time and study the movements, interactions and roles of individual molecules. For instance, they can identify and track the proteins that allow a virus to invade a cell or those that enable tumor cells to migrate to distant parts of the body in metastatic cancer. The ability to analyze individual molecules, rather than collections of molecules, allows scientists to answer longstanding questions about cellular mechanisms and behavior, such as how cells move along a surface or how certain proteins interact with DNA to regulate gene activity. Continue reading

Correcting a Cellular Routing Error Could Treat Rare Kidney Disease

AGT protein and peroxisomes in untreated and treated cells.
The altered AGT protein (red) and peroxisomes (green) appear in different places in untreated cells (top), but they appear together (shown in yellow) in cells treated with DECA (bottom). Credit: Carla Koehler/Reproduced with permission from Proceedings of the National Academy of Sciences USA. View larger image.

Our cells have organized systems to route newly created proteins to the places where they’re needed to do their jobs. For some people born with a rare and potentially fatal genetic kidney disorder called PH1, a genetically altered form of a particular protein mistakenly ends up in mitochondria instead of in another organelle, the peroxisome. This cellular routing error of the AGT protein results in the harmful buildup of oxalate, which leads to kidney failure and other problems at an early age.

In new work led by UCLA biochemist Carla Koehler Exit icon, researchers used a robotic screening system to identify a compound that interferes with the delivery of proteins to mitochondria. Koehler’s team Exit icon showed that adding a small amount of the compound, known as DECA, to cells grown in the laboratory prevented the altered form of the AGT protein from going to the mitochondria and sent it to the peroxisome. The compound also reduced oxalate levels in a cell model of PH1.

The team’s findings suggest that DECA, which is already approved by the Food and Drug Administration for treating certain bacterial infections, could be a promising candidate for treating children affected by PH1. And, Koehler notes, the screening strategy that she and her team used to identify DECA as a potential therapy may help researchers identify other new therapies for the disorder.

This work was funded in part by NIH under grant R01GM061721.

Molecules Known to Damage Cells May Also Have Healing Power

Free radicals in an ying-yang symbol
Biology in balance: Molecules called free radicals—like the peroxide molecules illustrated here—have a reputation for being dangerous. Now, they’ve revealed healing powers. In worms, at least. Credit: Stock image

When our health is concerned, some molecules are widely labeled “good,” while others are considered “bad.” Often, the truth is more complicated.

Consider free radical molecules. These highly reactive, oxygen-containing molecules are well known for damaging DNA, proteins and other molecules in our bodies. They are suspected of contributing to premature aging and cancer. But now, new research shows they might also have healing powers Exit icon.

Using the oft-studied laboratory roundworm known as C. elegans, a research group led by Andrew Chisholm Exit icon at the University of California, San Diego, made a surprising discovery. Free radicals, specifically those made in cell structures called mitochondria, appear necessary for skin wounds to heal. In fact, higher (but not dangerously high) levels of the molecules can actually speed wound closure.

If further research shows the same holds true in humans, the work could point to new ways to treat hard-to-heal wounds, like diabetic foot ulcers.

This work was funded in part by NIH under grants R01GM054657 and P40OD010440.

Cool Image: Snap-Together Laboratory

Modular microfluidics system

Modular microfluidics system. Credit: University of Southern California Viterbi School of Engineering.

Like snapping Lego blocks together to build a fanciful space station, scientists have developed a new way to assemble a microfluidics system, a sophisticated laboratory tool for manipulating small volumes of fluids.

Microfluidics systems are used by scientists to perform tasks as diverse as DNA analysis, microbe detection and disease diagnosis. Traditionally, they have been slow and expensive to produce, as each individual “lab on a chip” had to be built from scratch in a special facility.

Now, researchers including Noah Malmstadt Exit icon of the University of Southern California have harnessed 3-D printing technology to create a faster, cheaper, easier-to-use system Exit icon. The team first identified the smallest functional pieces of a microfluidics system. Each of these pieces performs one simple task like detecting the size of fluid droplets or mixing two fluids together. After 3-D printing individual components, the team showed that they could be snapped together by hand into a working system in a matter of hours. The individual pieces can be pulled apart and re-assembled as needed before use in an actual experiment, which was impossible with the traditional microfluidics systems.

The researchers have created eight block-like components so far. They hope to start an online community where scientists will share designs for additional components in an open-source database, helping to speed further development of the technology.

This work was funded in part by NIH under grant R01GM093279.

Meet Jennifer Doudna

Jennifer Doudna
Credit: Jennifer Doudna
Jennifer Doudna
Fields: Biochemistry and structural biology
Studies: New genome editing tool called CRISPR
Works at: University of California, Berkeley
Raised in: Hilo, Hawaii
Studied at: Pomona College, Harvard University
Recent honors: Winner of the Lurie Prize in the Biomedical Sciences Exit icon, an annual award that recognizes outstanding achievement by promising scientists age 52 or younger
If she couldn’t be a scientist, she’d like to be: A papaya farmer or an architect

Jennifer Doudna likes to get her hands dirty. Literally. When she’s not in her laboratory, she can often be found amid glossy green leaves and brightly colored fruit in her Berkeley garden. She recently harvested her first three strawberry guavas.

Coaxing tropical fruit plants from her childhood home in Hawaii to grow in Northern California is more than just a hobby—it’s an intellectual challenge.

“I like solving puzzles, I like the process of figuring things out, and I enjoy working with my hands,” says Doudna. “Those things were what really drew me to science in the beginning.”

Since she was a graduate student, Doudna’s professional puzzle has been RNA, a type of genetic material inside our cells. Recently, there has been an explosion of discoveries about the many roles RNA molecules play in the body. Doudna’s work probes into how RNA molecules work, what 3-D shapes they form and how their structures drive their functions.

“I’ve been fascinated by understanding RNA at a mechanistic level,” Doudna says.

While teasing out answers to these fundamental questions, Doudna’s lab has played a leading role in a discovery that is upending the field of genetic engineering, with exciting implications for human health.

Her Findings

The discovery started with bacteriophages—viruses that infect bacteria, just like the common cold infects humans. About 10 years ago, researchers using high-powered computing to sift through bacterial genomes began to find mysterious repetitive gene sequences that matched those from viruses known to infect the bacteria. The researchers named these sequences “clustered regularly interspaced short palindromic repeats,” or CRISPRs for short.

Over the next few years, scientists came to understand that these CRISPR sequences are part of something not previously thought to exist—an adaptive bacterial immune system, which remembers viruses fought off before and raises a response to fight them when exposed again. CRISPRs were this immune system’s reference library, holding records of viral exposure.

Somehow, bacteria with a CRISPR-based immune system (there are three types now known to scientists) use these records to command certain proteins to recognize and chop up DNA from returning viruses.

Wanting to know more about this process, Doudna’s team picked one protein in a CRISPR-based defense system to study. This protein, called Cas9, had been identified by other researchers as being essential for protection against viral invasion.

To their delight, Doudna’s group had hit the jackpot. Cas9 turned out to be the system’s scalpel. Once CRISPR identifies a DNA sequence from the invading virus, Cas9 slices the sequence out of the viral genome, destroying the virus’s ability to copy itself.

Doudna’s lab and their European collaborators also identified the other key components of the CRISPR-Cas9 system—two RNA molecules that guide Cas9 to the piece of viral DNA identified by CRISPR.

Even more importantly, the researchers showed that the two guide RNAs could be manipulated in the lab to create a tool that both recognizes any specified DNA sequence and carries Cas9 there to make its cut.

“That was really where we made the connection between the basic, curiosity-driven research that we were doing and recognizing that we had in our hands something that could be a very powerful technology for genome editing,” remembers Doudna.

She was right. After publication of their 2012 paper, the field of CRISPR-guided genetic manipulation exploded. Labs around the world now use the tool Doudna’s team developed to cut target gene sequences in organisms ranging from plants to humans. The technique is already replacing more time-consuming, less-reliable methods of creating ‘knock-out’ model organisms (those missing a specific gene) for laboratory research. CRISPR-based editing even allows more than one gene to be knocked out at the same time, something that was not possible with previous genome-editing techniques.

The ability of CRISPR systems to recognize DNA sequences with extraordinary precision also holds potential for human therapeutics. For example, a paper from another laboratory published early this year showed that, in a mouse model, CRISPR-based editing could cut out and replace a defective gene responsible for a type of muscular dystrophy. Researchers are testing similar CRISPR-based techniques in models of human diseases ranging from cystic fibrosis to blood disorders.

Doudna is a co-founder of two biotechnology companies hoping to harness the potential of CRISPR-based genome editing. Although the technology holds great promise, she acknowledges that much work needs to be done before CRISPR can be considered safe for human trials. Major challenges include assuring that no off-target cuts are made in the genome and finding a safe way to deliver the editing system to living tissues.

She is also excited to continue working with her research team, advancing the basic understanding of the CRISPR-based system.

“I’m very interested in seeing what we can contribute to the whole question about how you deliver a technology like this, how you can use it therapeutically in an organism. That’s an area where we hope that our biochemical understanding of this system will be able to contribute,” she concludes.

Modifying Bacterial Behavior

Biofilm
Communication through quorum sensing is key to the formation of biofilms, slimy bacterial communities that can cause infections and are often stubbornly resistant to antibiotics. Credit: P. Singh and E. Peter Greenberg.

Like a person trailing the aroma of perfume or cologne, bacteria emit chemical signals that let other bacteria of the same species know they’re there. Bacteria use this chemical communication system, called quorum sensing, to assess their own population size. When they sense a large enough group, or quorum, the microbes modify their behavior accordingly. Many disease-causing bacteria use quorum sensing to launch a coordinated attack when they’ve amassed in sufficient numbers to overwhelm the host’s immune response.

Chemist Helen Blackwell of the University of Wisconsin-Madison has been making artificial compounds that mimic natural quorum-sensing signals, as well as some that block a natural signal from binding to its protein target—a step needed to produce a change in bacterial behavior. By altering key building blocks in these protein targets one by one, Blackwell’s team found that small changes could convert an activation signal into an inhibitory signal, or vice versa, indicating that small-molecule control of quorum sensing is very finely tuned.

Improved understanding of the molecular basis of quorum sensing could help scientists design more potent compounds to disrupt these signals. Using such compounds to quiet quorum sensing may provide a new way to control disease-causing bacteria that reduces the chances an infection will become resistant to treatment.

This work was funded in part by NIH under grants R01GM109403 and T32GM008505.

Learn more:
University of Wisconsin-Madison News Release Exit icon
Blackwell Lab Exit icon
Learning From Bacterial Chatter Article from Inside Life Science
Bugging the Bugs Article from Findings Magazine

The Sweet Side of Chemistry

Glycans
Simple sugars connect in long, branched structures to create glycans. Credit: Stock image.

We’re in the middle of National Chemistry Week Exit icon, which this year focuses on “The Sweet Side of Chemistry—Candy.” Studying sugar chemistry is also relevant to our health.

The sugar in chocolate, taffy and other confections is a type of simple sugar called sucrose. In our bodies, sugars can exist in many forms, ranging from individual units like glucose to long, branched chains known as glycans containing thousands of individual sugar units linked together.

Glycans are involved in just about every aspect of how our cells work. They help make sure proteins are folded into the proper shape so they function correctly. They act as ZIP codes that direct newly made proteins to the right cellular locations. Some divert white blood cells to infection sites, and others serve as anchors for viruses to latch onto.

Because of the diverse and critical roles that glycans play in our bodies, chemists want to learn more about these molecules, with a long-term goal of harnessing them to treat or prevent disease. Read about some of their discoveries in the Why Sugars Might Surprise You article from Inside Life Science and the Life is Sweet article from Findings magazine. The You Are What You Eat chapter from ChemHealthWeb offers more details about the chemistry of sugar.

Nobel Prize for Powerful Microscopy Technology

Fibroblasts
The cells shown here are fibroblasts, one of the most common cells in mammalian connective tissue. These particular cells were taken from a mouse. Scientists used them to test the power of a new microscopy technique that offers vivid views of the inside of a cell. The DNA within the nucleus (blue), mitochondria (green) and cellular skeleton (red) is clearly visible. Credit: Dylan Burnette and Jennifer Lippincott-Schwartz, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.

William E. Moerner was at a conference in Brazil when he learned he’d be getting a Nobel Prize in chemistry. “I was incredibly excited and thrilled,” he said of his initial reaction.

An NIGMS grantee at Stanford University, Moerner received the honor for his role in achieving what was once thought impossible—developing super-resolution fluorescence microscopy, which is so powerful it allows researchers to see and track individual molecules in living organisms in real time.

Nobel recipients usually learn of the prize via a phone call from Stockholm, Sweden, in early October. For those in the United States, the call typically comes between 2:30 a.m. and 5:45 a.m.

Every year, the NIGMS communications office prepares for the Nobel Prize announcements in physiology or medicine and chemistry, the categories in which our grantees are most likely to be recognized. If the Institute played a significant role in funding the prize-winning research, we work quickly to provide information and context to reporters covering the story on tight deadlines. We issue a statement, identify an in-house expert on the research and arrange interviews with reporters. It’s all to help get the word out about the research and the taxpayers’ role in supporting it.

This year’s in-house expert, Cathy Lewis, shared her thoughts on the prize to Moerner in an NIGMS Feedback Loop post. You can also read this year’s statement and see a full list of NIGMS-supported Nobel laureates.

Outwitting Antibiotic Resistance

Marine scene with fish and corals
The ocean is a rich source of microbes that could yield infection-fighting natural molecules. Credit: National Oceanic and Atmospheric Administration Exit icon.

Antibiotics save countless lives and are among the most commonly prescribed drugs. But the bacteria and other microbes they’re designed to eradicate can evolve ways to evade the drugs. This antibiotic resistance, which is on the rise due to an array of factors, can make certain infections difficult—and sometimes impossible—to treat.

Read the Inside Life Science article to learn how scientists are working to combat antibiotic resistance, from efforts to discover potential new antibiotics to studies seeking more effective ways of using existing ones.