Dual-channel biological function generator

Rice University bioengineers who specialize in creating tools for synthetic biology have unveiled the latest version of their “biofunction generator and “bioscilloscope,” an optogenetic platform that uses light to activate and study two biological circuits at a time.

The biofunction generator and bioscilloscope are a toolkit of genes and hardware that use colored lights and engineered bacteria to bring both mathematical predictability and cut-and-paste simplicity to the world of genetic circuit design.

“Unfortunately, all biological light sensors are ‘sloppy,’ in that they tend to respond to multiple colors of light,” said Jeffrey Tabor, an associate professor of bioengineering at Rice. “We’ve developed a detailed mathematical model to capture this sloppiness and design multicolor light signals that compensate for it so that two light sensors can be independently controlled in the same cell. Because most of the circuits that control biological behaviors are composed of two or more genes, this technology will make it easier for our lab and others to study complex synthetic biological systems.”

The research is described in a recent paper in Molecular Systems Biology.

Life is controlled by DNA-based circuits. These are similar to the circuits in smartphones and other electronic devices with a key difference: The information that flows through electronic circuitry is voltage, and the information that flows through genetic circuits is protein production. Genetic circuits can be switched on or off — produce protein or not — and they can be tuned to produce more or less protein, much like voltage from an electronic circuit can be raised or lowered.

The biofunction generator and bioscilloscope, which were first created in Tabor’s lab three years ago, show how closely the analogy holds. Function generators and oscilloscopes, stock components of electrical engineering labs for more than 50 years, are test instruments that can feed voltage signals into circuits and show how signal voltage varies with time at other locations within the circuit. Oscilloscope screens usually show wave functions and can plot one or more signals at a time.

The bioscilloscope plots the output of biocircuits in exactly the same way. The inputs and outputs for the biocircuits are light. Specifically, Tabor’s team has developed a biofunction generator, a set of light-activated genes that can be used to turn genes on and off and to regulate the amount of protein they produce when turned on. The bioscilloscope comprises another set of genes that add fluorescent tags to the DNA to read out the circuit response, which means the more protein that’s produced, the more light that’s given off by the sample.

In the new paper, recent Ph.D. graduate and lead author Evan Olson and colleagues tested new dual-function tools using the latest optogenetic hardware and software tools developed by Tabor’s lab in conjunction with a new mathematical model for the biofunction generator output.

“The model allows us to predict the output gene-expression response to any light input signal, regardless of how the intensity or spectral composition of the light signal changes over time,” Olson said. “The model works by describing how light of any wavelength and intensity is converted into a population of light sensors in the ‘on’ or ‘off’ states.”

Olson said they demonstrated the system in two proof-of-concept experiments. In the first, they showed the system could compensate for “perturbative” signals, incoming light such as that from a microscope or fluorescent imager that might otherwise interfere with the incoming optogenetic signal. In the second, they demonstrated multiplexed control by simultaneously driving two independent gene expression signals in two optogenetic circuits in the same bacteria. The output on the bioscilloscope shows the two functions as red and green lines. The researchers showed they could activate the genetic circuits to produce smooth waves and stair-step patterns, and they showed the two circuits could be switched on in unison or at different times.

“This multiplexing approach enables a completely new generation of experiments for characterizing and controlling the biological circuits that integrate multiple signals and that are ubiquitous in biological networks, particularly those used for decision-making and developmental processes,” Tabor said.

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Materials provided by Rice University. Original written by Jade Boyd. Note: Content may be edited for style and length.

Scientists engineer baker’s yeast to produce penicillin molecules

The synthetic biologists from Imperial College London have re-engineered yeast cells to manufacture the nonribosomal peptide antibiotic penicillin. In laboratory experiments, they were able to demonstrate that this yeast had antibacterial properties against streptococcus bacteria.

The authors of the study, which is published in the journal Nature Communications, say their new method demonstrates the effectiveness of using this kind of synthetic biology as a route for discovering new antibiotics. This could open up possibilities for using re-engineered yeast cells to develop new forms of antibiotics and anti-inflammatory drugs from the nonribosomal peptide family.

Nonribosomal peptides are normally produced by bacteria and fungi, forming the basis of most antibiotics today. Pharmaceutical companies have long experimented with nonribosomal peptides to make conventional antibiotics. The rise of antimicrobial resistance means there is a need to use genetic engineering techniques to find a new range of antibiotics from bacteria and fungi. However, genetically engineering the more exotic fungi and bacteria- the ones likely to have antibacterial properties — is challenging because scientists don’t have the right tools and they are difficult to grow in a lab environment, requiring special conditions.

Baker’s yeast on the other hand is easy to genetically engineer. Scientists can simply insert DNA from bacteria and fungi into yeast to carry out experiments, offering a viable new host for antibiotic production research. The rise of synthetic biology methods for yeast will allow researchers to make and test many new gene combinations that could produce a whole new range of new antibiotics.

However, the authors are keen to point out that the research is still in its early stages. While this approach does show promise, they have so far produced nonribosomal peptide antibiotic penicillin in small quantities. More research needs to be done to see if it can be adapted to finding other compounds and to get production up to commercially viable quantities.

Dr Tom Ellis, from the Centre for Synthetic Biology at Imperial College London, explains: “Humans have been experimenting with yeast for thousands of years. From brewing beer to getting our bread to rise, and more recently for making compounds like anti-malarial drugs, yeast is the microscopic workhorse behind many processes.

“The rise of drug-resistant superbugs has brought a real urgency to our search for new antibiotics. Our experiments show that yeast can be engineered to produce a well-known antibiotic. This opens up the possibility of using yeast to explore the largely untapped treasure trove of compounds in the nonribosomal peptide family to develop a new generation of antibiotics and anti-inflammatories.”

Previously, scientists have demonstrated that they could re-engineer a different yeast to make penicillin. However, that species of yeast is not as well understood or amenable to genetic manipulation compared to baker’s yeast, used by the authors in today’s study, making it less suitable for the development of novel antibiotics using synthetic biology.

In their experiments, the team used genes from the filamentous fungus, from which nonribosomal peptide penicillin is naturally derived. These genes caused the yeast cells to produce the nonribosomal peptide penicillin via a two-step biochemical reaction process. First the cells made the nonribosomal peptide base — the ‘backbone’ molecule — by a complex reaction, and then this was modified by a set of further fungal enzymes that turn it into the active antibiotic.

During the experimentation process, the team discovered that they didn’t need to extract the penicillin molecules from inside the yeast cell. Instead, the cell was expelling the molecules directly into the solution it was in. This meant that the team simply had to add the solution to a petri-dish containing streptococcus bacteria to observe its effectiveness. In the future, this approach could greatly simplify the molecule testing and manufacturing process.

Dr Ali Awan, co-author from the Department of Bioengineering at Imperial College London, explains: “Fungi have had millions of years to evolve the capability to produce bacteria-killing penicillin. We scientists have only been working with yeast in this context for a handful of years, but now that we’ve developed the blueprint for coaxing yeast to make penicillin, we are confident we can further refine this method to create novel drugs in the future.

“We believe yeast could be the new mini-factories of the future, helping us to experiment with new compounds in the nonribosomal peptide family to develop drugs that counter antimicrobial resistance.”

The team are currently looking for fresh sources of funding and new industrial collaborators to take their research to the next level.

Dr Ellis added: “Penicillin was first discovered by Sir Alexander Fleming at St Mary’s Hospital Medical School, which is now part of Imperial. He also predicted the rise of antibiotic resistance soon after making his discovery. We hope, in some small way, to build on his legacy, collaborating with industry and academia to develop the next generation of antibiotics using synthetic biology techniques.”

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Materials provided by Imperial College London. Original written by Colin Smith. Note: Content may be edited for style and length.

First EPA-approved outdoor field trial for genetically engineered algae

Scientists at the University of California San Diego and Sapphire Energy have successfully completed the first outdoor field trial sanctioned by the U.S. Environmental Protection Agency for genetically engineered algae.

In a series of experiments funded by the U.S. Department of Energy, the researchers tested a genetically engineered strain of algae in outdoor ponds under real-world conditions. As reported in the journal Algal Research, the researchers conclude that genetically engineered algae can be successfully cultivated outdoors while maintaining engineered traits, and, most importantly, without adversely impacting native algae populations.

“Just as agricultural experts for decades have used targeted genetic engineering to produce robust food crops that provide human food security, this study is the first step to demonstrate that we can do the same with genetically engineered algae,” said Stephen Mayfield, a professor of biology and an algae geneticist at UC San Diego.

Under the EPA’s purview over a 50-day experiment, the scientists cultured strains of the algae species Acutodesmus dimorphus — genetically engineered with genes for fatty acid biosynthesis and green fluorescent protein expression — in parallel with non-engineered algal species. Testing both algae strains in water samples taken from five regional lakes showed strikingly similar levels of growth in the tests, and that the genetic modification did not change the impact of the cultivated strains on native algae communities.

“This study showed the framework for how this type of testing can be done in the future,” said study coauthor Jonathan Shurin, an ecologist in UC San Diego’s Division of Biological Sciences. “If we are going to maintain our standard of living in the future we are going to need sustainable food and energy, and ways of making those that do not disrupt the environment. Molecular biology and biotechnology are powerful tools to help us achieve that. Our experiment was a first-step towards an evidence-based evaluation of genetically engineered algae and their benefits and environmental risks.”

“Progress made in the lab means little if you can’t reproduce the phenotype in a production setting,” said Shawn Szyjka, the study’s lead author, formerly of Sapphire Energy.

Future testing will include additional gene types in experiments that run several months, allowing the researchers to further evaluate influences from weather, seasonal shifts and other environmental factors.

“Algae biomass can address many needs that are key to a sustainable future,” said Mayfield, director of the California Center for Algae Biotechnology and the Food and Fuel for the 21st Century initiative. “This is the first of many studies testing this technology in field settings.”

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Materials provided by University of California – San Diego. Original written by Mario Aguilera. Note: Content may be edited for style and length.

New capabilities for genome-wide engineering of yeast

One of humankind’s oldest industrial partners is yeast, a familiar microbe that enabled early societies to brew beer and leaven bread and empowers modern ones to synthesize biofuels and conduct key biomedical research. Yeast remains a vital biological agent, yet our ability to explore and influence its genomic activity has lagged.

In a new article in Nature Communications, University of Illinois researchers describe how their successful integration of several cutting-edge technologies — creation of standardized genetic components, implementation of customizable genome editing tools, and large-scale automation of molecular biology laboratory tasks — will enhance our ability to work with yeast. The results of their new method demonstrate its potential to produce valuable novel strains of yeast for industrial use, as well as to reveal a more sophisticated understanding of the yeast genome.

“The goal of the work was really to develop a genome-scale engineering tool for yeast . . . traditional metabolic engineering focused on just a few genes and the few existing genome-scale engineering tools are only applicable to bacteria, not eukaryotic organisms like yeast,” said Steven L. Miller Chair of Chemical and Biomolecular Engineering Huimin Zhao, who led the study. “A second innovation is the use of synthetic biology concepts, the modularization of the parts, and integration with a robotic system, so we can do it in high-throughput.”

The team focused on yeast in part because of its important modern-day applications; yeasts are used to convert the sugars of biomass feedstocks into biofuels such as ethanol and industrial chemicals such as lactic acid, or to break down organic pollutants. Because yeast and other fungi, like humans, are eukaryotes, organisms with a compartmentalized cellular structure and complex mechanisms for control of their gene activity, study of yeast genome function is also a key component of biomedical research.

“In basic science, a lot of fundamental eukaryotic biology is studied in yeast,” said Tong Si, a Carl R. Woese Institute for Genomic Biology Research Fellow. “People have a limited understanding of these complicated systems. Although there are approximately 6,000 genes in yeast, people probably know less than 1,000 by their functions; all the others, people do not know.”

The group took the first step toward their goal of a novel engineering strategy for yeast by creating what is known as a cDNA library: a collection of over 90% of the genes from the genome of baker’s yeast (Saccharomyces cerevisiae), arranged within a custom segment of DNA so that each gene will be, in one version, overactive within a yeast cell, and in a second version, reduced in activity.

Zhao and colleagues examined the ability of the CRISPR-Cas system, a set of molecules borrowed from a form of immune system in bacteria (CRISPR stands for clustered regularly interspaced short palindromic repeats, describing a feature of this system in bacterial genomes). This system allowed Zhao to make precise cuts in the yeast genome, into which the standardized genetic parts from their library could insert themselves.

“The first time we did this, in 2013, there was no CRISPR . . . the best we could get was 1% of the cells modified in one run,” said Si. “We struggled a little on that, and when CRISPR came out, that worked. We got it to 70% [cells modified], so that was very important.”

With gene activity-modulating parts integrating into the genome with such high efficiency, the researchers were able to randomly generate many different strains of yeast, each with its own unique set of modifications. These strains were subjected to artificial selection processes to identify those that had desirable traits, such as the ability to survive exposure to reagents used in the biofuel production process.

This selection process was greatly aided by the Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB), a robotic system that performs most of the laboratory work described above in an automated way, including selection of promising yeast strains. Use of iBioFAB greatly accelerated the work, enabling simultaneous creation and testing of many unique strains. The iBioFAB was conceived and developed by the Biosystems Design research theme at the Carl R. Woese Institute for Genomic Biology (IGB), which is led by Zhao.

With support from the High Performance Biological Computing Group at Illinois, Zhao, Si and their colleagues analyzed the modified genomes of their most promising yeast strains. They identified combinations of genes whose altered activities contributed to desirable traits; the functions of some of these genes were previously unknown, demonstrating the technique’s ability to generate new biological knowledge.

“I think the key difference between this method and the other existing metabolic engineering strategies in yeast is really the scale,” said Zhao. “The current metabolic engineering strategies are all focused on just a few genes, dozens of genes at most . . . it’s very intuitive. With this we can explore all the genes, we can identify a lot of targets that cannot be intuited.”