Unusual soybean coloration sheds a light on gene silencing

Today’s soybeans are typically golden yellow, with a tiny blackish mark where they attach to the pod. In a field of millions of beans, nearly all of them will have this look. Occasionally, however, a bean will turn up half-black, with a saddle pattern similar to a black-eyed pea.

“The yellow color is derived from a natural process known as gene silencing, in which genes interact to turn off certain traits,” explains Lila Vodkin, professor emerita in the Department of Crop Sciences at the University of Illinois. “Scientists make use of this process frequently to design everything from improved crops to medicines, but examples of naturally occurring gene silencing — also known as RNA interference, or RNAi — are limited. A better understanding of this process can explain how you can manipulate genes in anything from soybeans to humans.”

The RNAi pathway was discovered about 20 years ago as a naturally occurring process in a tiny roundworm. The discovery and follow-up work showing its biomedical potential won scientists the Nobel Prize in 2006. In plants, RNAi was discovered in petunias. When breeders tried to transform one gene to cause brighter pinks and purples, they wound up with white flowers instead. The gene for flower color had been silenced.

“Before they were domesticated, soybeans were black or brown due to the different anthocyanin pigments in the seed coat,” says Sarah Jones, a research specialist working with Vodkin on the study. “Breeders got rid of the dark pigments because they can discolor the oil or soybean meal during extraction processes.”

Vodkin clarifies, “The yellow color was a naturally occurring RNAi mutation that happened spontaneously, probably at the beginning of agriculture, like 10,000 years ago. People saw the yellow beans as different. They picked them up, saved them, and cultivated them. In the germplasm collections of the wild soybean, Glycine sojae, you don’t find the yellow color, only darkly pigmented seeds.”

Previous work from the team showed that yellow soybeans result from a naturally occurring gene silencing process involving two genes. Essentially, one of the genes blocks production of the darker pigment’s precursors. But the researchers weren’t sure why black pigments sometimes reappear, as in saddle-patterned beans. Now they know.

Vodkin and her team searched for beans with unusual pigmentation in the USDA soybean germplasm collection, housed at U of I. The collection contains thousands of specimens, representing much of the genetic diversity in domesticated soybean and its wild relatives.

“We requested beans with this black saddle pattern,” Jones recalls. “We wanted to know if they all get this pattern from the same gene.” Some of the samples had been collected as far back as 1945.

The team used modern genomic sequencing techniques, quickly sifting through some 56,000 protein-coding genes to identify the one responsible for the pattern. The lead author, Young Cho, made the discovery as a graduate student when he noticed a defect in the Argonaute5 gene. The team looked at additional beans with the saddle and found that the Argonaute5 gene was defective in a slightly different way in each of them.

“That’s how you prove you found the right gene,” Vodkin says, “because of these independent mutations happening at different spots right in that same gene.”

When the Argonaute5 gene is defective, the silencing process — which normally blocks the dark pigment and results in yellow beans — can no longer be carried out. The gene defect explains why the dark pigments show up in the saddle beans.

Before the team’s discovery, there were very few examples of how gene interactions work to achieve silencing in naturally occurring systems. Today, bioengineers use genetic engineering technologies to silence genes to produce a desired outcome, whether it’s flower color, disease resistance, improved photosynthesis, or any number of novel applications.

“The yellow color in soybeans could have been engineered, if it hadn’t occurred naturally,” Vodkin says, “but it would have cost millions of dollars and every yellow soybean would be a genetically modified organism. Nature engineered it first.” She says this study also emphasizes the value of the soybean germplasm collection, which preserves diversity for research and breeding purposes.

DNA delivery technology joins battle against drug-resistant bacteria

Antimicrobial resistance is one of the biggest threats to global health, affecting anyone, at any age, in any country, according to the World Health Organization. Currently, 700,000 deaths each year are attributed to antimicrobial resistance, a figure which could increase to 10 million a year by 2050 save further intervention.

New breakthrough technology from Tel Aviv University facilitates DNA delivery into drug-resistant bacterial pathogens, enabling their manipulation. The research expands the range of bacteriophages, which are the primary tool for introducing DNA into pathogenic bacteria to neutralize their lethal activity. A single type of bacteriophage can be adapted to a wide range of bacteria, an innovation which will likely accelerate the development of potential drugs based on this principle.

Prof. Udi Qimron of the Department of Clinical Microbiology and Immunology at TAU’s Sackler Faculty of Medicine led the research team, which also included Dr. Ido Yosef, Dr. Moran Goren, Rea Globus and Shahar Molshanski, all of Prof. Qimron’s lab. The study was recently published in Molecular Cell and featured on its cover.

For the research, the team genetically engineered bacteriophages to contain the desired DNA rather than their own genome. They also designed combinations of nanoparticles from different bacteriophages, resulting in hybrids that are able to recognize new bacteria, including pathogenic bacteria. The researchers further used directed evolution to select hybrid particles able to transfer DNA with optimal efficiency.

“DNA manipulation of pathogens includes sensitization to antibiotics, killing of pathogens, disabling pathogens’ virulence factors and more,” Prof. Qimron said. “We’ve developed a technology that significantly expands DNA delivery into bacterial pathogens. This may indeed be a milestone, because it opens up many opportunities for DNA manipulations of bacteria that were impossible to accomplish before.

“This could pave the way to changing the human microbiome — the combined genetic material of the microorganisms in humans — by replacing virulent bacteria with a-virulent bacteria and replacing antibiotic-resistant bacteria with antibiotic-sensitive bacteria, as well as changing environmental pathogens,” Prof. Qimron continued.

“We have applied for a patent on this technology and are developing products that would use this technology to deliver DNA into bacterial pathogens, rendering them a-virulent and sensitive to antibiotics,” Prof. Qimron said.

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E. coli bacteria's defense secret revealed

By tagging a cell’s proteins with fluorescent beacons, Cornell researchers have found out how E. coli bacteria defend themselves against antibiotics and other poisons. Probably not good news for the bacteria.

When undesirable molecules show up, the bacterial cell opens a tunnel though its cell wall and “effluxes,” or pumps out, the intruders.

“Dynamic assembly of these tunnels has long been hypothesized,” said Peng Chen, professor of chemistry and chemical biology. “Now we see them.”

The findings could lead to ways to combat antibiotic-resistant bacteria with a “cocktail” of drugs, he suggests: “One is to inhibit the assembly of the tunnel, the next is to kill the bacteria.”

To study bacteria’s defensive process, Chen and colleagues at Cornell selected a strain of E. coli known to pump out copper atoms that would otherwise poison the bacteria. The researchers genetically engineered it, adding to the DNA that codes for a defensive protein an additional DNA sequence that codes for a fluorescent molecule.

Under a powerful microscope, they exposed a bacterial cell to an environment containing copper atoms and periodically zapped the cell with an infrared laser to induce fluorescence. Following the blinking lights, they had a “movie” showing where the tagged protein traveled in the cell. They further genetically engineered the various proteins to turn their metal-binding capability on and off, and observed the effects.

Their research was reported in the Early Online edition of the Proceedings of the National Academy of Sciences the week of June 12. The Cornell researchers also collaborated with scientists at the University of Houston, the University of Arizona and the University of California, Los Angeles.

The key protein, known as CusB, resides in the periplasm, the space between the inner and outer membranes that make up the bacteria’s cell wall. When CusB binds to an intruder — in this experiment, a copper atom — that has passed through the porous outer membrane, it changes its shape so that it will attach itself between two related proteins in the inner and outer membranes to form a complex known as CusCBA that acts as a tunnel through the cell wall. The inner protein has a mechanism to grab the intruder and push it through.

The tunnel locks the inner and outer membranes together, making the periplasm less flexible and interfering with its normal functions. The ability to assemble the tunnel only when needed, rather than having it permanently in place, gives the cell an advantage, the researchers point out.

This mechanism for defending against toxic metals may also explain how bacteria develop resistance to antibiotics, by mutating their defensive proteins to recognize them. Similar mechanisms may be found in other species of bacteria, the researchers suggested.

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Materials provided by Cornell University. Original written by Bill Steele. Note: Content may be edited for style and length.

Highly safe biocontainment strategy hopes to encourage greater use of GMOs

Use of genetically modified organisms (GMOs) — microorganisms not found in the natural world but developed in labs for their beneficial characteristics — is a contentious issue.

For while GMOs could greatly improve society in numerous ways — e.g. attacking diseased cells, digesting pollution, or increasing food production — their use is heavily restricted by decades-old legislation, for fear of what might happen should they escape into the environment.

For researchers, aware of their potential, it is important to develop safety strategies to convince legislators they are safe for release.

For this reason Hiroshima University’s Professor Ryuichi Hirota and Professor Akio Kuroda, have developed an extra safe phosphite-based biocontainment strategy.

Biocontainment strategies — methods used to prevent GMO escape or proliferation beyond their required use, typically employ one of two forms.

One is “suicide switch” where released GMOs die off independently after a given time. The other is “nutrient requirement,” where GMOs are designed to expire on removal of a nutrient source.

The control method for the new genetically modified E. coli strain of bacteria employs the latter, and its simple practicality could prove a real game changer.

It relies on the fact that all living things require phosphorus for a vast array of life-determining processes including energy storage, DNA production, and cell signal-transduction. The overwhelming majority of bacteria, source phosphorus from the naturally occurring nutrient phosphate.

However, bacteria are renowned for their ability to obtain energy from seemingly implausible sources and the researchers at HU found one type, Ralstonia sp. Strain 4506, capable of utilizing non-naturally occurring phosphite instead — throwing up exciting possibilities.

As Phosphite, a waste by-product from the metal plating industry, does not occur in the natural world, scientists can easily control its availability and determine potential GMO proliferation.

Strain 4506’s phosphite-digesting enzyme was thus isolated and introduced into E. coli bacteria, which due to its versatility is considered the poster boy of the GMO world. Genetic editing also saw a phosphite specific “transporter” created to allow this nutrients intake.

But while this modified E. coli, now with phosphite munching capabilities, was quite the novelty in the HU lab, there was still a major hurdle to overcome — it still possessed innate phosphate transport mechanisms intact, and could equally survive on non-naturally occurring phosphite or naturally occurring phosphate. It could easily escape and thrive.

As E. coli has seven phosphate transporters — pumps for transferring phosphate from outside the cells membrane to inside, Professors Hirota and Kuroda set about shutting them down using genetic editing.

When the resulting GMO was tested the results were outstanding. It proliferated in a phosphite medium, and didn’t grow at all when exposed only to phosphate.

Further, when thriving populations were later deprived of their phosphite-hit, their numbers tumbled over a two week period to zero — thus fulfilling the criteria for “nutrition requirement” biocontainment.

However, what the scientists discovered next astounded them. Even when this new GMO was successfully and continuously cultured on phosphite, its population nevertheless still began plummeting after two weeks.

Baffled, the HU researchers are investigating but there is a possibility that this strategy possesses “suicide switch” characteristics on top of “nutrient requirement.”

Whatever the reason, an extremely safe and practical biocontainment strategy has been born. Requiring just nine simple gene edits, in naturally occurring organisms, and based on phosphite — a readily available industrial waste product; it is extremely cost and time effective. Additionally, its simplicity means it can be adapted for other microorganisms, making it highly versatile.

These traits contrast with previous biocontainment strategies involving synthetic organisms and energy sources, requiring hundreds of gene edits, awful lots of money and time, and which are so specialized as to make them impractical.

It is hoped this new strategy will grab the attention of relevant government agencies, and convince them to bring 1980s laws in line with 21st Century advancements. We can then get GMOs safely out of the lab for the betterment of society!

Bio-based p-xylene oxidation into terephthalic acid by engineered E. coli

KAIST researchers have established an efficient biocatalytic system to produce terephthalic acid (TPA) from p-xylene (pX). It will allow this industrially important bulk chemical to be made available in a more environmentally-friendly manner.

The research team developed metabolically engineered Escherichia coli (E. coli) to biologically transform pX into TPA, a chemical necessary in the manufacturing of polyethylene terephthalate (PET). This biocatalysis system represents a greener and more efficient alternative to the traditional chemical methods for TPA production. This research, headed by Distinguished Professor Sang Yup Lee, was published in Nature Communications on May 31.

The research team utilized a metabolic engineering and synthetic biology approach to develop a recombinant microorganism that can oxidize pX into TPA using microbial fermentation. TPA is a globally important chemical commodity for manufacturing PET. It can be applied to manufacture plastic bottles, clothing fibers, films, and many other products. Currently, TPA is produced from pX oxidation through an industrially well-known chemical process (with a typical TPA yield of over 95 mol%), which shows, however, such drawbacks as intensive energy requirements at high temperatures and pressure, usage of heavy metal catalysts, and the unavoidable byproduct formation of 4-carboxybenzaldehyde.

The research team designed and constructed a synthetic metabolic pathway by incorporating the upper xylene degradation pathway of Pseudomonas putida F1 and the lower p-toluene sulfonate pathway of Comamonas testosteroni T-2, which successfully produced TPA from pX in small-scale cultures, with the formation of p-toluate (pTA) as the major byproduct. The team further optimized the pathway gene expression levels by using a synthetic biology toolkit, which gave the final engineered E. coli strain showing increased TPA production and the complete elimination of the byproduct.

Using this best-performing strain, the team designed an elegant two-phase (aqueous/organic) fermentation system for TPA production on a larger scale, where pX was supplied in the organic phase. Through a number of optimization steps, the team ultimately achieved production of 13.3 g TPA from 8.8 g pX, which represented an extraordinary yield of 97 mol%.

The team has developed a microbial biotechnology application which is reportedly the first successful example of the bio-based production of TPA from pX by the microbial fermentation of engineered E. coli. This bio-based TPA technology presents several advantages such as ambient reaction temperature and pressure, no use of heavy metals or other toxic chemicals, the removable of byproduct formation, and it is 100% environmentally compatible.

Professor Lee said, “We presented promising biotechnology for producing large amounts of the commodity chemical TPA, which is used for PET manufacturing, through metabolically engineered gut bacterium. Our research is meaningful in that it demonstrates the feasibility of the biotechnological production of bulk chemicals, and if reproducible when up-scaled, it will represent a breakthrough in hydrocarbon bioconversions.”

Remembrance of things past: bacterial memory of gut inflammation

The microbiome, or the collections of microorganisms present in the body, is known to affect human health and disease and researchers are thinking about new ways to use them as next-generation diagnostics and therapeutics. Today bacteria from the normal microbiome are already being used in their modified or attenuated form in probiotics and cancer therapy. Scientists exploit the microorganisms’ natural ability to sense and respond to environmental- and disease-related stimuli and the ease of engineering new functions into them. This is particularly beneficial in chronic inflammatory diseases like inflammatory bowel disease (IBD) that remain difficult to monitor non-invasively. However, there are several challenges associated with developing living diagnostics and therapeutics including generating robust sensors that do not crash and are capable of long-term monitoring of biomolecules.

In order to use bacteria of the microbiome as biomarker sensors, their genome needs to be modified with synthetic genetic circuits, or a set of genes that work together to achieve a sensory or response function. Some of these genetic alterations may weaken or break normal signaling circuits and be toxic to these bacteria. Even in cases where the probiotic microbes tolerate the changes, the engineered cells can have growth delays and be outcompeted by other components of the microbiome. As a result, probiotic bacteria and engineered therapeutic microbes are rapidly cleared from the body, which makes them inadequate for long period monitoring and modulation of the organism’s tissue environment.

A team at the Wyss Institute of Biologically Inspired Engineering led by Pamela Silver, Ph.D., designed a powerful bacterial sensor with a stable gene circuit in a colonizing bacterial strain that can record gut inflammation for six months in mice. This study offers a solution to previous challenges associated with living diagnostics and may bring them closer to use in human patients. The findings are reported in Nature Biotechnology.

Silver, who is a Core Faculty member at the Wyss Institute and also the Elliot T. and Onie H. Adams Professor of Biochemistry and Systems Biology at Harvard Medical School, thought of the gut as a first application for this system due to its inaccessibility by non-invasive means and its susceptibility to inflammation in patients suffering from chronic diseases like IBD. “We think about the gut as a black box where it is hard to see, but we can use bacteria to illuminate these dark places. There is great interest from patients and doctors that push us to build sensors for biomarkers of gut conditions like IBD and colon cancer,” said Silver, “We believe that our work opens up enormous possibilities that can exploit the flexibility and modularity of our diagnostic tool and expand the use of engineered organisms to a wide variety of applications.”

Key to the team’s work is the introduction of a memory module to the circuit that is able to detect a molecule of interest and respond to this exposure long after the stimulus is gone. As bacteria can be rapidly cleared from the intestinal tract, the team used a strain of bacteria that is part of the microbiome of mice, and engineered it to contain the sensory and memory elements capable of detecting tetrathionate. Tetrathionate is a transient metabolic molecule produced in the inflamed mouse intestine as a result of either infection with pathogenic bacteria like Salmonella typhimurium and Yersinia enterocolitica or genetic defects affecting inflammation.

The synthetic genetic circuit designed by the Wyss team contains a “trigger element” that is adopted from the natural system specifically recognizing the biomarker (in this case tetrathionate) in cells, or that can be developed using synthetic approaches when no prior sensor exists. The second element in the circuit is the “memory element” that resembles a toggle switch and has been adapted from a virus that attacks bacteria. It consists of two genes (A and B for simplicity) that regulate each other depending on whether the stimulus is present. In the tetrathionate sensor, the product of gene A blocks expression of gene B when tetrathionate is absent. When tetrathionate is produced during inflammation and is sensed by the trigger element, levels of A decrease and the gene B is induced and begins to shut off expression of gene A. The expression of the B gene is also coupled to a reporter gene which turns bacteria from colorless to blue only when they have switched the memory element on. The switch can be maintained in the on state long after the first tetrathionate exposure.

After verifying the functionality of the sensor in a liquid culture of bacteria, David Riglar, Ph.D., the study’s first author, was able to show that it detected tetrathionate in a mouse model of gut inflammation caused by infection with S. typhimurium up to six months after administration of the sensor-containing probiotic bacteria. Through simple analysis of fecal matter, the synthetic circuit’s memory state was confirmed to be on and its DNA unchanged and stable. “Our approach is to use the bacteria’s sensing ability to monitor the environment in unhealthy tissue or organs. By adding gene circuits that retain memory, we envision giving humans probiotics that record disease progression by a simple and non-invasive fecal test,” said Riglar.

Silver’s team plans to extend this work to sensing inflammation in the human gut and also to develop new sensors detecting signs of a variety of other conditions.

“Pam’s work demonstrates the power of synthetic biology for advancing medicine as it provides a way to rationally and rapidly design sophisticated sensors for virtually any molecule. If successful in humans, their technology would offer a much less expensive and more specific way to monitor gut function at home than sophisticated imaging instruments used today,” said Donald Ingber, M.D., Ph.D., Founding Director of the Wyss Institute, the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

Scientists borrow from electronics to build circuits in living cells

Living cells must constantly process information to keep track of the changing world around them and arrive at an appropriate response.

Through billions of years of trial and error, evolution has arrived at a mode of information processing at the cellular level. In the microchips that run our computers, information processing capabilities reduce data to unambiguous zeros and ones. In cells, it’s not that simple. DNA, proteins, lipids and sugars are arranged in complex and compartmentalized structures.

But scientists — who want to harness the potential of cells as living computers that can respond to disease, efficiently produce biofuels or develop plant-based chemicals — don’t want to wait for evolution to craft their desired cellular system.

In a new paper published May 25 in Nature Communications, a team of UW synthetic biology researchers have demonstrated a new method for digital information processing in living cells, analogous to the logic gates used in electric circuits. They built a set of synthetic genes that function in cells like NOR gates, commonly used in electronics, which each take two inputs and only pass on a positive signal if both inputs are negative. NOR gates are functionally complete, meaning one can assemble them in different arrangements to make any kind of information processing circuit.

The UW engineers did all this using DNA instead of silicon and solder, and inside yeast cells instead of at an electronics workbench. The circuits the researchers built are the largest ever published to date in eurkaryotic cells, which, like human cells, contain a nucleus and other structures that enable complex behaviors.

“While implementing simple programs in cells will never rival the speed or accuracy of computation in silicon, genetic programs can interact with the cell’s environment directly,” said senior author and UW electrical engineering professor Eric Klavins. “For example, reprogrammed cells in a patient could make targeted, therapeutic decisions in the most relevant tissues, obviating the need for complex diagnostics and broad spectrum approaches to treatment.”

Each cellular NOR gate consists of a gene with three programmable stretches of DNA — two to act as inputs, and one to be the output. The authors then took advantage of a relatively new technology known as CRISPR-Cas9 to target those specific DNA sequences inside a cell. The Cas9 protein acts like a molecular gatekeeper in the circuit, sitting on the DNA and determining if a particular gate will be active or not.

If a gate is active, it expresses a signal that directs the Cas9 to deactivate another gate within the circuit. In this way, the researchers can “wire” together the gates to create logical programs in the cell.

What sets the study apart from previous work, researchers said, is the scale and complexity of the circuits successfully assembled — which included up to seven NOR gates assembled in series or parallel.

At this size, circuits can begin to execute really useful behaviors by taking in information from different environmental sensors and performing calculations to decide on the correct response. Imagined applications include engineered immune cells that can sense and respond to cancer markers or cellular biosensors that can easily diagnose infectious disease in patient tissue.

These large DNA circuits inside cells are a major step toward an ability to program living cells, the researchers said. They provide a framework where logical programs can be easily implemented to control cellular function and state.

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Materials provided by University of Washington. Note: Content may be edited for style and length.