Microorganisms possess natural product biosynthetic gene clusters (BGCs) that may harbor unique bioactivities for use in drug development and agricultural applications. However, many uncharacterized microbial BGCs remain inaccessible. Researchers at Illinois previously demonstrated a technique using transcription factor decoys to activate large, silent BGCs in bacteria to aid in natural product discovery.
Now, they have developed a direct cloning method that aims to accelerate large-scale discovery of novel natural products. Their findings are reported in the journal Nature Communications.
Named Cas12a assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE), the method allows for direct cloning of large genomic fragments, including those with high-GC content or sequence repeats. Where existing direct cloning methods fail to effectively clone natural product BGCs of this nature, CAPTURE excels.
“Using CAPTURE, microbial natural product BGCs can be directly cloned and heterologously expressed at an unprecedented rate,” said study leader and Steven L. Miller Chair professor of chemical and biomolecular engineering Huimin Zhao (BSD leader/CABBI/GSE/MMG). “As a result, CAPTURE allows large-scale cloning of natural product BGCs from various organisms, which can lead to discovery of numerous novel natural products.”
Researchers first characterized the efficiency and robustness of CAPTURE by cloning 47 natural product BGCs from both Actinomycetes and Bacilli. After demonstrating nearly 100% efficiency of CAPTURE, 43 uncharacterized natural product BGCs from 14 Streptomyces and three Bacillus species were cloned and heterologously expressed by researchers. The produced compounds were purified and determined as 15 novel natural products, including six unprecedented compounds designated as bipentaromycins. Four of the bipentaromycins exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Bacillus anthracis.
“Addressing the current antimicrobial resistance crisis requires discovery of novel molecules capable of treating drug-resistant infections,” said Zhao. “Discovery of bipentaromycins not only demonstrates the possibility of discovering novel antimicrobials, but it also provides an example on how this strategy can be applied for discovery of unique bioactive compounds for use in drug development and agricultural applications.”
The researchers plan next to characterize these compounds for other bioactivities such as anticancer, antiparasitic and anticancer properties. Preliminary results are already showing anticancer properties for some of the compounds.
“Due to its exceptional robustness and efficiency, CAPTURE will likely become the method of choice for direct cloning of large DNA molecules such as natural product BGCs from genomic or metagenomic DNA for various basic and applied biological applications,” said Zhao.
Researchers used single-molecule imaging to compare the genome-editing tools CRISPR-Cas9 and TALEN. Their experiments revealed that TALEN is up to five times more efficient than CRISPR-Cas9 in parts of the genome, called heterochromatin, that are densely packed. Fragile X syndrome, sickle cell anemia, beta-thalassemia and other diseases are the result of genetic defects in the heterochromatin.
The researchers report their findings in the journal Nature Communications.
The study adds to the evidence that a broader selection of genome-editing tools is needed to target all parts of the genome, said Huimin Zhao, a professor of chemical and biomolecular engineering at the University of Illinois Urbana-Champaign who led the new research.
“CRISPR is a very powerful tool that led to a revolution in genetic engineering,” Zhao said. “But it still has some limitations.”
CRISPR is a bacterial molecule that detects invading viruses. It can carry one of several enzymes, such as Cas-9, that allow it to cut viral genomes at specific sites. TALEN also scans DNA to find and target specific genes. Both CRISPR and TALEN can be engineered to target specific genes to fight disease, improve crop plant characteristics or for other applications.
Zhao and his colleagues used single-molecule fluorescence microscopy to directly observe how the two genome-editing tools performed in living mammalian cells. Fluorescent-labeled tags enabled the researchers to measure how long it took CRISPR and TALEN to move along the DNA and to detect and cut target sites.
“We found that CRISPR works better in the less-tightly wound regions of the genome, but TALEN can access those genes in the heterochromatin region better than CRISPR,” Zhao said. “We also saw that TALEN can have higher editing efficiency than CRISPR. It can cut the DNA and then make changes more efficiently than CRISPR.”
TALEN was as much as five times more efficient than CRISPR in multiple experiments.
The findings will lead to improved approaches for targeting various parts of the genome, Zhao said.
“Either we can use TALEN for certain applications, or we could try to make CRISPR work better in the heterochromatin,” he said.
The National Institutes of Health and National Science Foundation support this work.
Zhao is a member of the Carl R. Woese Institute for Genomic Biology at the U. of I.
Diana Yates | Life Sciences Editor, U. of I. News Bureau
The paper “TALEN outperforms Cas9 in editing heterochromatin target sites” is available from the U. of I. News Bureau.
Researchers have identified key ingredients for producing high-value chemical compounds in an environmentally friendly fashion: repurposed enzymes, curiosity, and a little bit of light.
A paper published in Nature describes a study led by Xiaoqiang Huang (pictured), a postdoctoral researcher in the University of Illinois at Urbana-Champaign’s Department of Chemical and Biomolecular Engineering (ChBE) and the Carl R. Woese Institute for Genomic Biology (IGB). Huang works in the lab of ChBE Professor Huimin Zhao, Conversion Theme Leader at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a U.S. Department of Energy-funded Bioenergy Research Center (BRC).
Catalysts are substances used to speed up chemical reactions; in living organisms, protein molecules called enzymes catalyze reactions in a process called biocatalysis.
Biocatalysis is rapidly emerging as a nuanced, agile way to synthesize valuable compounds. Scientists are investigating the ability of enzymes to catalyze diverse reactions, and for good reason: biocatalytic reactions are highly selective, meaning that scientists can use enzymes to act on specific substrates and create target products.
Enzymatic reactions are also highly sustainable as they are relatively inexpensive, consume low levels of energy, and do minimal damage to the environment: while chemical catalysts typically require organic solvents, heat, and high pressure to function, biocatalysts work in aqueous solutions, operating at room-temperature and normal-pressure conditions.
Despite their value to science and sustainability, enzymes can be complicated to work with. Reactions enzymes can catalyze are limited to those found in nature; this means that scientists often struggle to track down the perfect biocatalyst to meet their need.
The process is similar to mixing paint: How can an artist creatively combine the colors already on a palette to produce the right shade? In the language of a chemical reaction: How can scientists leverage enzymes already existing in nature to create the products they need?
The research team developed a solution: a visible-light-induced reaction that uses the enzyme family ene-reductase (ER) as a biocatalyst and can produce high yields of valuable chiral carbonyl compounds.
Scientists engineering valuable microbes for renewable fuels and bioproducts have developed a fast, efficient way to identify the most promising varieties.
Researchers at the University of Illinois at Urbana-Champaign developed a novel mass spectrometry-based screening technique to rapidly profile medium-chain fatty acids produced in yeast — part of a larger group of free fatty acids that are key components in essential nutrients, soaps, industrial chemicals, and fuels. They also identified seven new genetically engineered mutants of the yeast Saccharomyces cerevisiae that produce higher levels of those fatty acids.
The study is detailed in a paper published in the journal Biotechnology and Bioengineering. The research was performed at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a U.S. Department of Energy-funded Bioenergy Research Center, by two U of I professors: Huimin Zhao, Professor of Chemical and Biomolecular Engineering (ChBE), and Jonathan Sweedler, Professor of Chemistry and Director of the School of Chemical Sciences. The lead author is Pu Xue (above), a ChBE doctoral student with Zhao’s lab at CABBI.
Zhao’s group genetically engineers tiny yeast cells to increase production of fatty acids, crucial components of biodiesel, fatty alcohols, waxes and olefins — the building blocks for detergents, adhesives, and plastics. CABBI’s goal is to develop robust yeasts that can convert renewable plant biomass to fuels and chemicals, as an environmentally friendly and sustainable alternative to petroleum-based chemical manufacturing processes.
Scientists can create a large library of engineered yeast strains, or mutants, producing various MCFAs very quickly, Xue said. But their ability to control the exact composition of MCFAs produced in these microbial cell factories is limited, with no “high-throughput” way to quickly analyze large numbers of samples.
To overcome this limitation, Xue and other researchers worked with Sweedler’s group to develop a high-throughput screening tool, a chemical characterization approach based on MALDI-ToF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). Mass spectrometry is an information-rich way to analyze complex samples by measuring their mass-to-charge ratio of ions. A laser is shot at a colony of yeast on a slide; the instrument measures the molecular masses of the lipids in the yeast, and thus is able to identify them.
Most existing processes for detecting or analyzing free fatty acids rely on more complex gas or liquid chromatography-mass spectrometry, considered the gold standard methods. But those methods have limitations when dealing with a large pool of variations, and preparing samples is time-consuming and labor-intensive.
The overall approach using MALDI-ToF MS system is faster and has been used successfully with proteins and peptides. The ToF mass analyzer is fast, relatively low cost and has a large detection window, well-suited for screening complex biological targets.
But medium-chain fatty acids are highly volatile and lightweight, making them harder to detect and quantify with this approach. Moreover, the thick cell wall of yeast creates another challenge for researchers to decompose cellular compositions efficiently.
So the CABBI researchers optimized the sample preparation steps with various solvents and matrices. And instead of trying to directly detect the MCFAs, they used a proxy: membrane lipids in yeast cells. They hypothesized that increased levels of membrane lipids with shorter acyl chain phosphatidylcholines (PCs), a class of phospholipids, would correlate with a greater capacity to produce MCFAs, which are shorter in length than the abundant fatty acids commonly found in S. cerevisiae.
To validate that hypothesis, U of I scientists compared the MALDI-ToF MS profile of the naturally occurring yeast with two genetically engineered strains previously found to produce higher levels of MCFAs. The data showed that these two mutants had more of the shorter acyl chain PCs than the naturally occurring yeast. Those preliminary findings were then confirmed by the more exact liquid chromatography and fragment mass spectrometry processes.
With their established screening method in hand, CABBI team members set out to find more mutant strains with higher production of MCFAs. They found two prominent peaks on the mass spectrum that were correlated with the phospholipids, an indication of fatty acids. Those were then used as a sign of MCFA production.
As Sweedler points out, “the mass spectrometry measurement is fast — analyzing up to 2,000 yeast colonies per hour (approximately one sample every two seconds) compared to 30 minutes per sample under traditional methods.” The processing time is also significantly shortened: two to three minutes vs. three to four hours per sample. Overall, the MALDI-ToF MS screening tool allows scientists to quickly identify strains that warrant more detailed analysis.
“Our method allows us to screen tons of mutants in a short time. We can identify the good candidates for further study,” Xue said.
In the future, the method can be modified and used for high-throughput screening of other types of products, such as longer-chain fatty acids or fatty alcohols, saving time and labor. The researchers hope to build on their work at CABBI’s iBioFAB — the Illinois Biological Foundry for Advanced Biomanufacturing. Its robotic system can speed up sample preparation and provide faster, more accurate results, helping scale up the project to test many more mutant strains, Xue said. With about 6,000 genes in S. cerevisiae, millions of potential synergistic effects and features could be discovered.
Beyond science, these C6 to C12 fatty acids are important in human health, providing critical nutrients and useful products, such as Omega-3 fatty acids.
“In the future if we can directly generate biofuels and bioproducts such as fatty acids from microbial cells like yeast in large scale, that means we don’t need to use petrol,” Xue said. “We can save the environment and save a lot of money as well.”
Co-authors on the paper included CABBI researchers Shekhar Mishra (Chemical and Biomolecular Engineering) and Kisurb Choe (Biochemistry); former CABBI researcher Tong Si (ChBE); and Linzixuan Zhang, an undergraduate student in ChBE. All are also affiliated with the Carl R. Woese Institute for Genomic Biology at Illinois.
Professor and graduate students use 3D printer to supply emergency and healthcare workers
Several weeks ago, as the COVID-19 pandemic was spreading, Ying Diao, professor of Chemical and Biomolecular Engineering, and her research group, including several graduate students, began thinking about how they could help fight the outbreak.
Diao’s research group adopted the Montana Mask design, a 3D printable and reusable filtration mask with design files that are free for public use. The group has optimized its laboratory 3D printers to make 10 Montana Masks per day, with its goal to fabricate, assemble, and donate hundreds of masks to healthcare workers facing dire supply shortages.
Through an inspiring NPR story, Diao learned about the creation of 3D-printed ventilator parts in Italy. She immediately realized that her lab could potentially make facemasks and parts for medical supplies through their collective expertise in 3D printing (creating three-dimensional objects from computer-aided design models) and fabrication.
“I quickly found YouTube videos from 3D printing enthusiasts that have put up facemask designs using low cost, accessible materials,” Diao said. “So I picked the brain of my students on this idea and challenged my group to take this into action.”
They’ve already made an impact. The group just sent out its first shipment of 70 masks; 20 went to the Monticello Police Department and the Piatt County Sheriff’s Department, and 50 went to Parkland Memorial Hospital in Dallas, Texas. The group established Champaign County Covid Relief, where people can find updates, protocols, and links for resources to make their own printed or sewn masks.
They are still seeking recipients for masks that they’ve made, and they encourage healthcare workers and emergency first responders to contact them at email@example.com or Jadii Rodgers at firstname.lastname@example.org.
Along with Diao, the task force includes graduate students Bijal Patel, Prapti Kafle, Daniel Davies, and Zhuang Xu. All students are volunteering their time to create the masks, and Patel, the project lead, is running the two lab 3D printers for printing the Montana Masks and filter cartridges.
The Department of Chemical and Biomolecular Engineering donated $1,000 to help print the masks, and the group gratefully acknowledges support from Lori Sage-Karlson, a receiving manager in the School of Chemical Sciences, Jadii Rodgers, a departmental assistant in the Department of Chemical and Biomolecular Engineering, and others for their assistance in shipping masks, communicating with end users, and maintaining lab space during the pandemic.
Patel has been working heavily on 3D printing for his normal graduate research, so assembling the masks is fairly straightforward for him. Every 12 hours, he gathers what’s been printed, checks them, and hands them off to the assembly team. Then he starts a new set.
After printing the masks, the group sands them down for smoothness, scrubs them with soap and water, and sanitizes them in a bleach solution. Then, the assembly team attaches rubber weather stripping to form a seal, and adds elastic, filters, and other accessories before packaging them for shipment.
Kafle’s role in the mask production includes disinfecting, assembling components, and packaging. After sanding the masks and disinfecting them in bleach, she inserts the filter holder and strap and disinfects the parts again. The mask includes a furnace filter and a simple cloth filter.
The process of disinfecting and assembling the masks feels like performing an experiment, Kafle said. When she was given the opportunity to contribute to the effort, Kafle said she wanted to join in.
“When my advisor brought up the idea of using the equipment in our lab plus our skills and time to make the masks, I immediately wanted to be a part of it,” she said. “I had read numerous news about the shortage of masks among healthcare workers and that thousands of them across the globe are getting COVID-19.”
Along with Kafle, Davies also works on prototyping the designs and coming up with ways to make them better and more comfortable. The group recently received some elastic for tying on the masks; Davies said that it was easy to investigate which designs would fit their parameters because the 3D printing community is quite open and sharing.
“Since our lab is currently shut down for regular work, we had extra time to spend on new projects like this,” he said. “I have my own 3D printer at home, so I thought it would be a good idea to at least make masks for ourselves and family members, and it made prototyping pretty easy.”
The lab uses two 3D printers for producing optical and electronic materials.
“Initially what we were seeing was that it would be difficult to actually make PPE to the standards necessary to keep health care workers safe,” Davies said. “But the Montana mask seemed to be the best option. From there, Bijal worked with the local hospitals to make sure they were usable, and we have been making adjustments to make our masks as effective as possible.”
Patel said he’s been frustrated by the reported shortages of personal protective equipment for healthcare workers.
“Running this project and doing the best we can to help healthcare workers is a way to turn that frustration into something productive,” he said.
Researchers in the Schroeder and Moore groups at the University of Illinois at Urbana-Champaign have published a new study that illustrates how changes in the polymer sequence affect charge transport properties. This work required the ability to build and study chain molecules with high levels of precision.
The paper, “Charge Transport in Sequence-Defined Conjugated Oligomers,” was published in the Journal of the American Chemical Society.
Chain molecules or polymers are ubiquitous in modern society, with organic electronic materials increasingly used in solar cells, flat panel displays, and sensors. However, conventional materials are generally made by statistical polymerization, where the order of the subunits or monomers — the monomer sequence — is random.
“Traditional polymerization methods do not give us a perfect level of control of sequence,” said Charles Schroeder, the associate head and Ray and Beverly Mentzer Professor in Chemical and Biomolecular Engineering and a full-time faculty member at the Beckman Institute for Advanced Science and Technology. “As a result, it has been challenging to ask how the monomer sequence affects its properties.”
The researchers developed a method called iterative synthesis to deal with the problem. “Protein synthesis in our cells occurs by adding the amino acids one by one. We use the same method for making synthetic polymers where we add distinct monomers in a one-by-one fashion. This allows us to precisely control the sequence in a linear arrangement,” said Hao Yu, a graduate student in the Schroeder Group, and the Moore Group led by Jeff Moore, the Stanley O. Ikenberry Endowed Chair and professor of chemistry.
After making the materials, the researchers studied their charge transport properties using single molecule techniques. In this way, they were able to measure the conductance through single chains, much like a ‘molecular wire.’
“Molecular wires are generally good at transporting charge,” Schroeder said. “We wanted to know how the charge transport properties change if the overall sequence changes.”
Yu added molecular anchors at both ends of the chain molecule to enable the characterization. “We used a technique called the scanning tunneling microscope-break junction method, where the anchors link to two gold electrodes and form a molecular junction,” said Songsong Li, a graduate student in the Schroeder Group. “Then we impose an applied bias or voltage across the molecule, and this allows us to measure the charge transport properties of these polymers.”
“Currently the synthesis method is labor intensive,” Schroeder said. “Moving forward, we are developing automated synthesis methods in the Beckman Institute to generate large libraries of sequence-defined molecules.”
“The implications of this work are significant,” said Dawanne Poree, program manager at the Army Research Office that supports the work. “It’s often been wondered if the sequence-dependent properties observed in biological polymers could translate to synthetic polymeric materials. This work represents a step toward answering this question. Additionally, this work provides key insights into how molecular structure can be rationally designed and manipulated to render materials with designer properties of interest to the Army such as nanoelectronics, energy transport, molecular encoding, and data storage, self-healing, and more.”
The work was supported by the U.S. Department of Defense by a multidisciplinary university research initiative through the Army Research Office, an element of the U.S. Army Combat Capabilities Development Command’s Army Research Laboratory.
Editor’s note: The paper “Charge Transport in Sequence-Defined Conjugated Oligomers” can be found at https://doi.org/10.1021/jacs.0c00043.
University of Illinois researchers have developed a new method that aids in the process of making valuable compounds by using a unique combination of catalysts.
A study published this month in Nature reported a new method that combines enzymatic catalysts with photocatalysts.
The team that created the method was led by Professor Huimin Zhao, the Steven L. Miller Chair of Chemical and Biomolecular Engineering. Zhao is also Conversion Theme Leader at the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a U.S. Department of Energy-funded Bioenergy Research Center. Zhao worked alongside John Hartwig, Professor of Chemistry at the University of California at Berkeley, who was previously a professor at Illinois and is a longtime collaborator of Zhao’s. Coauthors include Yajie Wang, a Ph.D. Candidate in Chemical and Biomolecular Engineering at Illinois, and Zachary Litman, a Postdoctoral Research Fellow at the University of Michigan.
All organisms rely on chemical reactions in order to make various natural products. These reactions can be caused by a number of enzymatic or chemical catalysts.
Scientists often use combinations of enzymatic and chemical catalysts to cause reactions that result in higher yields than what can be achieved with enzymes alone. Higher yields are beneficial when scientists want to use these reactions to make useful products such as biofuels and pharmaceuticals.
But combining enzymatic and chemical catalysts is difficult to do — the two catalysts aren’t naturally compatible; they work best under different conditions and temperatures.
Seeing the need for new approaches, Wang and Litman came up with an idea: to combine enzymatic catalysis and photocatalysis.
Photocatalysis, which uses light to spur on a chemical reaction, is currently a popular research area.
“Lots of researchers have tried to develop new photocatalysts for different reactions,” Zhao said. “And enzyme catalysis is a relatively old field.”
While photocatalysis and enzyme catalysis have been studied separately, few researchers have put these two catalysts together.
The team studied several enzymes and photocatalysts and found a pair that works together.
“If you use a light-driven process instead of one driven by heat, then that will be compatible with the enzymes,” Hartwig said.
The scientists not only showed that enzymatic catalysts and photocatalysts can be combined, but that this combination can also be productive. The method proved useful in creating a few important active pharmaceutical intermediates (APIs) for producing pharmaceutical drugs.
“(This research) would be quite useful for synthetic chemistry,” Hartwig said.
Zhao said the team will pursue further research in this area as a part of CABBI, a collaboration between the Carl R. Woese Institute for Genomic Biology (IGB) and the Institute for Sustainability, Energy, and Environment (iSEE) along with 17 partner institutions.
One of CABBI’s goals is to engineer biological systems that produce non-natural compounds, which can be used for biofuels, jet fuels, lubricants, and more.
The researchers hope to find more combinations of photocatalysts and enzymatic catalysts for more chemical reactions.
“This is just a first example,” Zhao said. “We want to find more chemoenzymatic systems for different types of reactions.”
Zhao expects this research will also inspire new discoveries.
“There are many new photocatalysts for different types of reactions, and there are many enzymes,” he said. “So I can see that this will stimulate researchers to develop new systems that can combine these two types of catalysts for making a wide variety of useful compounds.”
As the global datasphere expands, so does the need for more data storage. Enter DNA, the carrier of life’s genetic information, which offers a potential storage media of unprecedented density, durability, and efficiency.
However, using DNA as storage is currently costly and there is a lack of processing systems suited for this technology.
The University of Illinois at Urbana-Champaign is undertaking a $1.5 million effort to produce new DNA-based storage nanoscale devices using chimeric DNA, a hybrid molecule made from two different sources. Chemical and Biomolecular Engineering Professor Charles Schroeder is one of the Illinois researchers collaborating on the project. As part of the three-year project, “SemiSynBio: An on-chip nanoscale storage system using chimeric DNA,” the team will design a method to read, write, and store data in a more cost-effective way than current DNA storage techniques.
“We’re going to take cheap, native DNA and combine it with chemically modified nucleotides,” said Olgica Milenkovic, Illinois professor of electrical and computer engineering and researcher in the Coordinated Science Lab. “It will be stored and accessed using a novel implementation of a state-of-the-art semiconductor system.”
By 2025, the world will generate 163 zettabytes (one trillion gigabytes) of data a year, in part due to emerging technologies such as the Internet of Things, predicts research group IDC. While not all of that data will be stored, the demand for storage may outpace traditional storage capabilities.
DNA is an attractive solution because it can store up to 455 exabytes (1 exabyte is 1 billion gigabytes) in one gram, according to a 2015 report in the New Scientist. In addition, it is remarkably durable.
“We are still finding bones from over 100,000 years ago from which we can extract DNA,” said Milenkovic. “We don’t know of another storage medium that has such durability.”
During the course of the project, the research team will design and synthesize hybrid DNA molecules that contain non-natural chemical modifications. The goal is to expand the storage capacity enabled by DNA coding methods, essentially increasing the number of equivalent bits.
To accomplish this task, the team will employ concepts from molecular design and engineering and chemical synthesis.
The goal is to create a device that mimics the capabilities of a computer’s hard drive.
“Synthesis of chemically modified DNA essentially constitutes the ‘write’ step, and development of new methods for processing and de-encoding the chemical information stored in the DNA will constitute the ‘read’ step,” said Charles Schroeder, Professor and the Ray and Beverly Mentzer Faculty Scholar in Chemical and Biomolecular Engineering at Illinois. “I envision that the ‘reading’ and ‘storage’ steps could be built into an integrated device.”
The grant is funded through SemiSynBio, a partnership between the National Science Foundation (NSF) and the Semiconductor Research Corporation (SRC), which seeks to lay the groundwork for future information storage systems at the intersection of biology, physics, chemistry, computer science, materials science and engineering. Altogether, SemiSynBio is funding eight projects for a total of $12 million.
In addition to Milenkovic and Schroeder, Illinois collaborators include Jean-Pierre Leburton, the Gregory E. Stillman Professor of Electrical and Computer Engineering and professor of Physics, and Xiuling Li, Professor of Electrical and Computer Engineering. Leburton is also a researcher in the Beckman Institute and the Coordinated Science Lab, while Li is part of the Micro and Nanotechnology Lab.
Milenkovic also recently received a three-year, $2.5 million grant from DARPA to combine synthetic DNA with computing. Her co-PIs include Huimin Zhao and Alvaro Hernandez of Illinois, David Soloveichik of the University of Texas at Austin, and Marc Riedel of the University of Minnesota.
Written by Kim Gudeman, Coordinated Science Lab
For more information about the SemiSynBio program, please see the NSF news release.
The polymers that make up synthetic materials need time to de-stress after processing, researchers said. A new study has found that entangled, long-chain polymers in solutions relax at two different rates, marking an advancement in fundamental polymer physics. The findings will provide a better understanding of the physical properties of polymeric materials and critical new insight to how individual polymer molecules respond to high-stress processing conditions.
The study, published in the journal Physical Review Letters, could help improve synthetic materials manufacturing and has applications in biology, mechanical and materials sciences as well as condensed matter physics.
“Our single-molecule experiments show that polymers like to show off their individualistic behavior, which has revealed unexpected and striking heterogeneous dynamics in entangled polymer solutions,” said co-author Charles Schroeder, a professor of chemical and biomolecular engineering and faculty member of the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign. “A main goal of our research is to understand how single polymers – acting as individuals – work together to give materials macroscopic properties such as viscosity and toughness.”
Using a technique called single-molecule fluorescence microscopy, researchers can watch – in real time – as individual polymer molecules relax after the stretching, pulling and squeezing of the manufacturing process. “Imagine looking into a bowl of cooked spaghetti and watching the motion of a single noodle as the bowl is mixed,” Schroeder said.
“We found that the polymers exhibit one of two distinct relaxation modes,” said co-author and graduate student Yuecheng (Peter) Zhou. “One group of polymers relaxed via a single decaying exponential rate and the other group showed a two-phase process. That second population undergoes a very quick initial retraction followed by a slow relaxation. The existence of two different molecular populations was unexpected and not predicted by classic theory.”
This study worked with high molecular-weight DNA because it serves as an ideal model of other types of synthetic organic polymers, the researchers said.
“We chose DNA as our model polymer because it is a very large molecule and the chains are big enough to image in our microscope,” Schroeder said. “They are also all the same weight, which provided us with a very clean, well-defined system for data analysis.”
The researchers found that the percentage of the molecular subpopulation that exhibits the two-phase relaxation behavior increases as the overall polymer concentration increases in the entangled solutions.
“We are not certain why the single-mode relaxation or fast-retraction mode seems to be concentration-dependent, but it may have to do with enhanced interpolymer friction – the more polymers, the higher the chance they will interact, especially out of equilibrium,” Zhou said. “We are working with theorists here at the University of Illinois to better explain the single-mode and two-mode relaxation phenomena.”
The team is excited to bring new insight to the understanding of how complex fluids flow and how they are processed and manufactured, especially with polymers that are subjected to intense stress, such as the fluids that are used for 3D printing.
The National Science Foundation supported this research.
Written by Lois Yoksoulian, University of Illinois News Bureau
To reach Charles Schroeder, email email@example.com.
The CRISPR-Cas9 system has given researchers the power to precisely edit selected genes. Now, researchers have used it to develop a technology that can target any gene in the yeast Saccharomyces cerevisiae and turn it off by deleting single letters from its DNA sequence.
Such genome-scale engineering – in contrast to traditional strategies that only target a single gene or a limited number of genes – allows researchers to study the role of each gene individually, as well as in combination with other genes. It also could be useful for industry, where S. cerevisiae is widely used to produce ethanol, industrial chemicals, lubricants, pharmaceuticals and more.
Understanding and optimizing the genome could create yeast strains with increased productivity, said study leader Huimin Zhao, the Steven L. Miller Chair of Chemical Engineering and a member of the Carl R. Woese Institute for Genomic Biology at the U. of I. Zhao’s group published the new findings in the journal Nature Biotechnology.
“We want to use microorganisms as cellular factories to make valuable chemicals and biofuels,” Zhao said. “The scale we have demonstrated in this study is unprecedented. CRISPR has been used to introduce point mutations – for example, to address genetic diseases – but Saccharomyces yeast has about 6,000 genes, and we want to be able to knock out each of these genes iteratively and find out how they affect the production of a target compound.”
Researchers produce “knockout” yeast – where one gene has been deleted, or “knocked out” – to study how each gene contributes to the function of the cell. When a beneficial mutation is found, they can selectively breed yeast with that characteristic. Leading methods to produce knockout yeast excise the entirety of the targeted gene. This creates unintended problems, Zhao said, because many genes overlap each other. Deleting one gene also deletes portions of others, affecting multiple functions and making it difficult for researchers to truly isolate the effects of a single gene.
Each letter in a DNA sequence corresponds to a base, the building blocks that make up DNA chains. Zhao’s group harnessed the precision of the CRISPR-Cas9 system to create a technique that allows them to delete just one base in a gene’s DNA sequence. Since a cell “reads” DNA three bases at a time, this shifts the reading frame and knocks out the gene. Genes that overlap with the edited one remain unchanged and functional.
“We can introduce just one single base change on the entire chromosome. That makes a minimal disturbance in the function of the neighboring genes, so we can study how important the gene is in its cellular context. That kind of precision has not been achieved before,” Zhao said.
Their technique, named CRISPR/Cas9 and homology-directed-repair assisted genome-scale engineering or CHAnGE, has the advantages of being quick, efficient and low-cost, in addition to its precision. Zhao’s group developed a library of knockout yeast, one for each gene in the S. cerevisiae genome, and are making it available to other researchers for a $50 handling fee.
“In the past, teams of people would spend several years trying to knock out every gene in a yeast. With CHAnGE, one person can generate a library of yeast mutants covering the entire genome in about a month,” Zhao said.
Zhao’s group is working to develop libraries for other types of yeast, including ones that produce lipids used in lubricants, biofuels and other industrial applications.
The U.S. Department of Energy and the Carl R. Woese Institute for Genomic Biology at the U. of I. supported this work. Zhao is also a professor in the Carle Illinois College of Medicine at Illinois.
By Liz Ahlberg Touchstone, University of Illinois News Bureau