Test Tube Artificial Neural Network Recognizes "Molecular Handwriting"

Conceptual illustration of a droplet containing an artificial neural network made of DNA that has been designed to recognize complex and noisy molecular information, represented as 'molecular handwriting.' Credit: Olivier Wyart

Qian Lab - "Winner Take All" Method Enables DNA Computing

Test tube chemistry using synthetic DNA molecules can be utilized in complex computing tasks to exhibit artificial intelligence

Researchers at Caltech have developed an artificial neural network made out of DNA that can solve a classic machine learning problem: correctly identifying handwritten numbers. The work is a significant step in demonstrating the capacity to program artificial intelligence into synthetic biomolecular circuits.

The work was done in the laboratory of Lulu Qian, assistant professor of bioengineering. A paper describing the research (paywall) appears online on July 4 and in the July 19 print issue of the journal Nature.

"Though scientists have only just begun to explore creating artificial intelligence in molecular machines, its potential is already undeniable," says Qian. "Similar to how electronic computers and smart phones have made humans more capable than a hundred years ago, artificial molecular machines could make all things made of molecules, perhaps including even paint and bandages, more capable and more responsive to the environment in the hundred years to come."

Artificial neural networks are mathematical models inspired by the human brain. Despite being much simplified compared to their biological counterparts, artificial neural networks function like networks of neurons and are capable of processing complex information. The Qian laboratory's ultimate goal for this work is to program intelligent behaviors (the ability to compute, make choices, and more) with artificial neural networks made out of DNA.

"Humans each have over 80 billion neurons in the brain, with which they make highly sophisticated decisions. Smaller animals such as roundworms can make simpler decisions using just a few hundred neurons. In this work, we have designed and created biochemical circuits that function like a small network of neurons to classify molecular information substantially more complex than previously possible," says Qian.

To illustrate the capability of DNA-based neural networks, Qian laboratory graduate student Kevin Cherry chose a task that is a classic challenge for electronic artificial neural networks: recognizing handwriting.

Human handwriting can vary widely, and so when a person scrutinizes a scribbled sequence of numbers, the brain performs complex computational tasks in order to identify them. Because it can be difficult even for humans to recognize others' sloppy handwriting, identifying handwritten numbers is a common test for programming intelligence into artificial neural networks. These networks must be "taught" how to recognize numbers, account for variations in handwriting, then compare an unknown number to their so-called memories and decide the number's identity.

WHY DNA?

Key to creating biomolecular circuits out of DNA are the strict binding rules between molecules of DNA. A single-stranded DNA molecule is composed of smaller molecules called nucleotides—abbreviated A, T, C, and G—arranged in a string, or sequence. The nucleotides in a single-stranded DNA molecule can bond with those of another single strand to form double-stranded DNA, but the nucleotides bind only in very specific ways: An A nucleotide with a T or a C nucleotide with a G.

Taking advantage of these predictable binding rules, Qian and her colleagues can design short strands of DNA to undergo predictable chemical reactions in a test tube and thereby compute tasks, such as molecular pattern recognition. In 2011, Qian and her colleagues created the first artificial neural network made of DNA molecules that could recognize four simple patterns.

In the work described in the Nature paper, Cherry, who is the first author on the paper, demonstrated that a neural network made out of carefully designed DNA sequences could carry out prescribed chemical reactions to accurately identify "molecular handwriting." Unlike visual handwriting that varies in geometrical shape, each example of molecular handwriting does not actually take the shape of a number. Instead, each molecular number is made up of 20 unique DNA strands chosen from 100 molecules, each assigned to represent an individual pixel in any 10 by 10 pattern. These DNA strands are mixed together in a test tube.

"The lack of geometry is not uncommon in natural molecular signatures yet still requires sophisticated biological neural networks to identify them: for example, a mixture of unique odor molecules comprises a smell," says Qian.

Given a particular example of molecular handwriting, the DNA neural network can classify it into up to nine categories, each representing one of the nine possible handwritten digits from 1 to 9.

First, Cherry built a DNA neural network to distinguish between handwritten 6s and 7s. He tested 36 handwritten numbers and the test tube neural network correctly identified all of them. His system theoretically has the capability of classifying over 12,000 handwritten 6s and 7s—90 percent of those numbers taken from a database of handwritten numbers used widely for machine learning—into the two possibilities.

Crucial to this process was encoding a "winner take all" competitive strategy using DNA molecules, developed by Qian and Cherry. In this strategy, a particular type of DNA molecule dubbed the annihilator was used to select a winner when determining the identity of an unknown number.

"The annihilator forms a complex with one molecule from one competitor and one molecule from a different competitor and reacts to form inert, unreactive species," says Cherry. "The annihilator quickly eats up all of the competitor molecules until only a single competitor species remains. The winning competitor is then restored to a high concentration and produces a fluorescent signal indicating the networks' decision." 

Next, Cherry built upon the principles of his first DNA neural network to develop one even more complex, one that could classify single digit numbers 1 through 9. When given an unknown number, this "smart soup" would undergo a series of reactions and output two fluorescent signals, for example, green and yellow to represent a 5, or green and red to represent a 9.

Qian and Cherry plan to develop artificial neural networks that can learn, forming "memories" from examples added to the test tube. This way, Qian says, the same smart soup can be trained to perform different tasks.

"Common medical diagnostics detect the presence of a few biomolecules, for example cholesterol or blood glucose." says Cherry. "Using more sophisticated biomolecular circuits like ours, diagnostic testing could one day include hundreds of biomolecules, with the analysis and response conducted directly in the molecular environment."

The paper is titled "Scaling up molecular pattern recognition with DNA-based winner-take-all neural networks." Funding was provided by the National Science Foundation, the Burroughs Wellcome Fund, and the Shurl and Kay Curci Foundation.

Related:

• Computing with Biochemical Circuits Made Easy
• Caltech Researchers Create the First Artificial Neural Network Out of DNA
• Caltech Researchers Build Largest Biochemical Circuit Out of Small Synthetic DNA Molecules

ORIGINAL: Caltech

by Lori Dajose

07/05/2018

Electric eel inspires bio-friendly power source, what happens next may shock you

Could a device inspired by the electric eel offer a safer way to power medical implants?

Scientists are always on the lookout for safer, more natural ways to power devices that go into our bodies. After all, who really needs toxic battery elements and replacement surgery?

One organism that is pretty good at generating biocompatible power (for itself, at least) is the electric eel, and scientists have now used the high-voltage species as a blueprint for a promising new self-charging device that could one day power things like pacemakers, prosthetics and even augmented reality contact lenses.

Electric eels generate voltage through long stacks of thin cells that run end-on-end through their bodies. Called electrocytes, these cells create electricity by allowing sodium ions to rush into one end and potassium ions out the other, all at the same time. The voltage created by each cell is small, but together, the stacks within a single eel can generate as many as 600 V.

To recreate this effect, researchers from the University of Fribourg, the University of Michigan and the University of California San Diego turned to the difference in salinity between fresh and saltwater. They deposited hydrogel, ion-conducting blobs onto clear plastic sheets and separated them with ion-selective membranes.

Hundreds of blobs containing salt and freshwater were arranged in an alternating pattern. When the team had all these gel compartments make contact with one another, they were able to generate 100 V through what is known as reverse electrodialysis, where energy is generated through differing salt concentrations in the water.

While the eel triggers the simultaneous contact of its electrocytes using a neurotransmitter called acetylcholine as the command signal, the team achieved this by carefully working a special origami pattern – called a Miura-ori fold – into the plastic sheet. This meant that when pressure was applied to the sheet, it quickly snapped together and the cells shifted into exactly the right positions to create the electricity.

The device, which the team calls an artificial electric organ, isn't in the same ball park as an eel in terms of output, but the researchers do have some ideas around how to boost its efficiency. It points to the metabolic energy created by ion differences in the eel's stomach, or the mechanical muscle energy, as some of the possibilities, but does note that recreating these would be a major challenge.

"The electric organs in eels are incredibly sophisticated, they're far better at generating power than we are," Mayer said. "But the important thing for us was to replicate the basics of what's happening."

The research was published in the journal Nature. You can hear from Mayer in the video below.

 

Source: University of Fribourg, University of Michigan

ORIGINAL: NewAtlas
Nick Lavars
December 14th, 2017

Squishy Clockwork Biobot Could Dose You With Drugs From the Inside

Photo: Sau Yin Chin. Soft and 3D-printed micromachines can be implanted in the body to deliver doses of a chemo drug.

When Swiss watchmakers invented the Geneva drive, a two-geared mechanism that produces precise ticks forward, they probably never imagined that bioengineers would one day craft a 15-millimeter version out of squishy hydrogel. But then, they weren’t trying to make a biocompatible micromachine that could be implanted in the body to deliver doses of drugs.

This strange new biobot comes from the lab of Samuel Sia, a professor of biomedical engineering at Columbia University, in New York City. It uses neither battery nor wires, and can be controlled from outside the body to deliver a dose on command. It’s a gadget well suited for this new era of personalized medicine, Sia tells IEEE Spectrum. “Doctors want to see how the patient is doing and then modify the therapy accordingly,” he says.

He has already tested the gizmo in lab mice with bone cancer, with exciting results that were published today in the journal Science Robotics. More on that experiment later.

Photos: Sau Yin Chin The researchers constructed their Geneva device layer by layer, in a process that took about 30 minutes.

Sia’s team first had to invent a type of 3D printing to fabricate their tiny Geneva drive and several other soft micromachines. They came up with a fabricator that lays down layers of a hydrogel to produce rubbery solid shapes. While human hands are required to put the pieces together, Sia says those assembly steps could be automated. And it’s pretty quick, as is: The whole process of printing and assembling one Geneva drive takes less than 30 minutes. Today’s typical 3D printers would take several hours to construct a similar device, Sia says, and most can’t handle soft materials like hydrogel.

Here’s the part that runs like clockwork! The squishy Geneva drive clicks forward when an external magnet moves a simple gear, which is just a rubbery piece with embedded iron nanoparticles (the black curved piece in the video below). With each click, one of six chambers lines up with a hole and a dose of medicine flows out. In the video, a magnet (the silver disk) keeps the device running continuously to demonstrate the mechanism, but in clinical use, a doctor could apply a magnet only when a dose is required.

You may be wondering: Could someone’s implanted micromachine be triggered accidentally by an external magnet or by a malicious person with fiendish magnetic powers? In other words, is the X-Men’s Magneto a risk factor? “Somebody walking by with a magnet won’t trigger it, but there are some cases where it’s not ideal,” Sia says. His lab is working on other ways to wirelessly drive the mechanism, including an ultrasound technique.

The hardest part of the design process was getting the material right, Sia says. Very flexible and soft materials are compatible with the body’s soft innards, unlike rigid silicon or metal devices. “But if your material is collapsing like jello, it’s hard to make robots out of it,” he says. “It has to be stiff enough to work like a tiny implantable machine.”

Image: Sau Yin Chin The pieces of the Geneva device were each printed in soft hydrogel.

The next step was in vivo.
Sia’s team wanted to see if their devices would work inside the body, with all the complications of chemistry and anatomy. Some mice with bone cancer received implanted devices that were loaded up with a chemo drug; other mice received typical chemotherapy, which floods the whole body with a toxic drug. When the team compared the effects of the device’s localized and periodic delivery of the drug to those of the typical treatment, the results were impressive. The bionic mice’s tumors grew slower, more tumor cells died off, and fewer cells elsewhere in the body suffered peripheral damage.

Photos: Sau Yin Chin Fluorescent imaging shows a chemo-delivering device inside a lab mouse.

The clinical possibilities seem obvious—oncologists could deliver more targeted and concentrated doses of powerful chemo drugs, and Sia imagines other uses, like regulating the release of hormones. But the drug delivery device is really just a proof of concept, he says. He’s not rushing out to form a startup: “We have to do the cost-benefit analysis to see if this is really a commercializable device,” he says.

He is bullish, however, on the medical potential of tiny squishy robots in general. Soft and mobile little bots could one day act as internal repair crews, doing a doctor’s work from the inside. (For more on this, check out IEEE Spectrum’s article on medical microbots.) Sia says his fabrication platform is capable of turning out a wide variety of devices. “I’m confident that we’ll find something useful,” he says.

Sia won’t say exactly what types of devices his lab is now experimenting with, except to say that they’re looking at implanted devices that move. Here’s my guess: It’s a tiny squishy micromachine that resembles a cuckoo clock.

ORIGINAL: IEEE Spectrum
By Eliza Strickland
4 Jan 2017

Bringing Silicon to Life

Scientists persuade nature to make silicon-carbon bonds

A new study is the first to show that living organisms can be persuaded to make silicon-carbon bonds—something only chemists had done before. Scientists at Caltech "bred" a bacterial protein to have the ability to make the man-made bonds, a finding that has applications in several industries.

Molecules with silicon-carbon, or organosilicon, compounds are found in pharmaceuticals as well as in many other products, including agricultural chemicals, paints, semiconductors, and computer and TV screens. Currently, these products are made synthetically, since the silicon-carbon bonds are not found in nature.

The new research, which recently won Caltech's Dow Sustainability Innovation Student Challenge Award (SISCA) grand prize, demonstrates that biology can instead be used to manufacture these bonds in ways that are more environmentally friendly and potentially much less expensive.

"We decided to get nature to do what only chemists could do—only better," says Frances Arnold, Caltech's Dick and Barbara Dickinson Professor of Chemical Engineering, Bioengineering and Biochemistry, and principal investigator of the new research, published in the Nov. 24 issue of the journal Science.

The study is also the first to show that nature can adapt to incorporate silicon into carbon-based molecules, the building blocks of life. Scientists have long wondered if life on Earth could have evolved to be based on silicon instead of carbon. Science-fiction authors likewise have imagined alien worlds with silicon-based life, like the lumpy Horta creatures portrayed in an episode of the 1960s TV series Star Trek. Carbon and silicon are chemically very similar. They both can form bonds to four atoms simultaneously, making them well suited to form the long chains of molecules found in life, such as proteins and DNA.

"No living organism is known to put silicon-carbon bonds together, even though silicon is so abundant, all around us, in rocks and all over the beach," says Jennifer Kan, a postdoctoral scholar in Arnold's lab and lead author of the new study. Silicon is the second most abundant element in Earth's crust.

The researchers used a method called directed evolution, pioneered by Arnold in the early 1990s, in which new and better enzymes are created in labs by artificial selection, similar to the way that breeders modify corn, cows, or cats. Enzymes are a class of proteins that catalyze, or facilitate, chemical reactions. The directed evolution process begins with an enzyme that scientists want to enhance. The DNA coding for the enzyme is mutated in more-or-less random ways, and the resulting enzymes are tested for a desired trait. The top-performing enzyme is then mutated again, and the process is repeated until an enzyme that performs much better than the original is created.

Directed evolution has been used for years to make enzymes for household products, like detergents; and for "green" sustainable routes to making pharmaceuticals, agricultural chemicals, and fuels.

In the new study, the goal was not just to improve an enzyme's biological function but to actually persuade it to do something that it had not done before. The researchers' first step was to find a suitable candidate, an enzyme showing potential for making the silicon-carbon bonds.

Bringing Silicon to Life: Scientists Persuade Nature to Make Silicon-Carbon Bonds

Researchers in Frances Arnold’s lab at Caltech have persuaded living organisms to make chemical bonds not found in nature. The finding may change how medicines and other chemicals are made in the future.

Credit: Caltech

"It's like breeding a racehorse," says Arnold, who is also the director of the Donna and Benjamin M. Rosen Bioengineering Center at Caltech. "A good breeder recognizes the inherent ability of a horse to become a racer and has to bring that out in successive generations. We just do it with proteins."

The ideal candidate turned out to be a protein from a bacterium that grows in hot springs in Iceland. That protein, called cytochrome c, normally shuttles electrons to other proteins, but the researchers found that it also happens to act like an enzyme to create silicon-carbon bonds at low levels. The scientists then mutated the DNA coding for that protein within a region that specifies an iron-containing portion of the protein thought to be responsible for its silicon-carbon bond-forming activity. Next, they tested these mutant enzymes for their ability to make organosilicon compounds better than the original.

After only three rounds, they had created an enzyme that can selectively make silicon-carbon bonds 15 times more efficiently than the best catalyst invented by chemists. Furthermore, the enzyme is highly selective, which means that it makes fewer unwanted byproducts that have to be chemically separated out.

"This iron-based, genetically encoded catalyst is nontoxic, cheaper, and easier to modify compared to other catalysts used in chemical synthesis," says Kan. "The new reaction can also be done at room temperature and in water."

The synthetic process for making silicon-carbon bonds often uses precious metals and toxic solvents, and requires extra processing to remove unwanted byproducts, all of which add to the cost of making these compounds.

As to the question of whether life can evolve to use silicon on its own, Arnold says that is up to nature. "This study shows how quickly nature can adapt to new challenges," she says. "The DNA-encoded catalytic machinery of the cell can rapidly learn to promote new chemical reactions when we provide new reagents and the appropriate incentive in the form of artificial selection. Nature could have done this herself if she cared to."

The Science paper, titled "Directed Evolution of Cytochrome c for Carbon-Silicon Bond Formation: Bringing Silicon to Life," is also authored by Russell Lewis and Kai Chen of Caltech. The research is funded by the National Science Foundation, the Caltech Innovation Initiative program, and the Jacobs Institute for Molecular Engineering for Medicine at Caltech.

ORIGINAL: CALTECH

Written by Whitney Clavin

11/24/2016

Contact: Whitney Clavin

(626) 395-1856

wclavin@caltech.edu

CALIFORNIA INSTITUTE OF TECHNOLOGY

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Site content Copyright © 2016 California Institute of Technology

Launched: A Synthetic Biology Factory for Making Weird New Organisms

Photo: Eliza Strickland
The automated lab at Ginkgo Bioworks enables a design-build-test cycle for creating novel organisms.

Raising glasses of genetically modified beer, the synthetic biologists at Ginkgo Bioworks celebrated the launch of a new automated lab last month. By applying engineering principles to biology, and with the help of some nifty robotic equipment, Ginkgo has created a factory for churning out exotic lifeforms, the likes of which have never before been seen on this planet. 

The home brew was an example of the potential applications of synthetic biology, a new field that builds on recent progress in genetic assembly methods. Scientists can now manufacture snippets of synthetic DNA and slip them into organisms, giving those critters strange new capabilities.

Photo: Ana-Maria Murphy-Teixidor
Guests at the launch party sampled
Ginkgo Bioworks's home brew.

For example, the brewer’s yeast used to make beer for the launch party had genes from an orange tree added to its own DNA. During the fermentation stage of the brewing process, those genes caused the yeast to produce valencene, an organic compound with a citrusy flavor. Speaking scientifically, it was delicious.

Ginkgo Bioworks, a hip young company based in South Boston, recently raised $100 million on the promise of finding many such useful applications for synthetic biology. And it used some of that cash to build Bioworks2, the company’s vast new lab that uses robotic systems to make an assembly line for organisms.

Ginkgo needs to make microbes on a grand scale in order to find those that can function as tiny biological factories. When a client comes in with a request for a custom-made organism, Gingko begins its bulk experimentation. Many of the altered organisms will be duds, but through cycles of iteration the bioengineers eventually devise a microbe that turns out the desirable product. The company is messing around with organisms that produce chemical ingredients for perfumes, beverages, pesticides, and laundry detergent. 

Ginkgo Bioworks’ business model centers on the microbes themselves, not the end products. “We’re not in the business of manufacturing chemicals, flavors, or fragrances,” explains Ginkgo creative director Christina Agapakis. “We specialize in the organsims, and we partner with our customers who will make the product.” Ginkgo licenses organisms to its customers, she says, and gets royalties if they’re used.

But building an organism to spec is no easy task. Genetics still isn’t well understood; there’s no universal catalog listing of genes that details what they all do. And biology is messy. Even if researchers know what a particular gene does in an orange tree, for example, when they add it to a yeast cell, it might interact with the native DNA in unexpected ways. If they’re adding several genes from different species to that yeast cell, things get even more complicated.

That’s why Ginkgo takes an engineering approach to biology, hewing to a rigorous design-build-test cycle. In this case, they’re designing, building, and testing living organisms.

Photo: Eliza StricklandAt the new Bioworks2 lab, Ginkgo's synthetic biologists can test out thousands of variants for a new organism.

The new lab’s extreme automation is critical to this approach, says Patrick Boyle, Ginkgo’s head of organism design. “In grad school, I might have taken my five best ideas and tried them out,” Boyle says. “Here we take our 1000 best ideas, try them all out, and see which works best.” 

So how does that design, build, and test cycle work in practice? Take Ginkgo’s first efforts in the perfume business as an example. Ginkgo is working with the French perfumier Robertet on a yeast that spits out rose oil. There’s a business case for making this microbe: Extracting traditional rose oil from rose petals is expensive, and high-end perfumiers look down on chemical substitutes. But adding the right genes from a rose plant to a yeast cell could make it produce the real oil, just in an untraditional way. 

Design: A yeast serves as the biological “chassis,” the base for the customized creature. Ginkgo designers then search the scientific literature, looking for genes that would cause a cell to produce useful enzymes. They’re looking for enzymes that can work within the yeast cell’s metabolic process; when they feed sugar to the yeast it should carry out chemical reactions that ultimately result in rose oil. 

They hunt for genes all across the biological kingdoms: “We ask, ‘How have different biological niches solved this biochemistry problem, and how can we adapt them to our purposes?’” says Boyle. They can combine genes from different organisms into metabolic pathways, but this requires scaled-up science. “If you have 100 possible enzymes that can serve as a step in a four-step pathway, that’s a lot of design space to explore,” Boyle says.

Build: Ginkgo outsources the actual manufacturing of synthetic DNA, ordering up batches from companies like Twist Bioscience and Gen9. Boyle says the company will receive 700 million base-pairs of synthetic DNA in the next year and a half, which represents half the world’s current market for synthetic DNA. A company like Ginkgo can only exist because the cost of manufacturing DNA has recently come down dramatically. 

When a batch of manufactured DNA arrives at Ginkgo, liquid-handling robots add the various snippets to yeast cells and let the cells grow and multiply. Creative director Agapakis says these robots are getting better and better. “During my PhD, I spent a lot of time moving tiny amounts of fluid around,” she says. “When we started Ginkgo, a lot of the robots looked like eight-armed grad students—there were a lot of pipettes.”

Image: Labcyte

A high-tech liquid-handling robot 

moves samples via sound waves.

Now Ginkgo has liquid-handling robots like the Echo 525, which moves nanoliters of liquid using targeted pulses of sound. The machine sends ultrasonic waves upward at a sample plate, focusing on just one of the plate’s 1536 tiny wells, and propels the droplet of liquid upward to a new vessel. It can move the contents of a plate in about 20 minutes. 

Test: Once Ginkgo has 1000 yeast variants containing different mashups of genes, it’s time to see if the cells are making their product: for example, rose oil. The researchers use mass spectrometry machines to break apart the cells and examine all the molecules inside. They check whether the yeast is producing the oil, of course, but also whether the yeast is healthy. Using some of the cell’s metabolic energy to produce rose oil could interfere with other processes and “change the total picture,” says Agapakis. 

Even when they’ve found a few yeast variants that seem to do a good job of cranking out oil, Ginkgo’s job isn’t done. The bioengineers still need to see whether the organism can make a product that’s truly useful to the customer. 

Boyle says that in the case of rose oil, they study each yeast to determine its overall “fragrance profile.” While a cell may be making certain useful fragrance molecules, it may be making other molecules that are distinctly not useful. “I like the fresh-baked bread smell, but it’s not great when you’re trying to sell a perfume,” Boyle says. “So how do we cut down on background fragrances?” The perfume, which could currently be called eau de baguette, is still a work in progress.

Perfume is just the beginning for this ambitious company. Agapakis sees biological manufacturing as the way of the future, and she doesn’t mind sounding like “a college student in a dorm room” when she talks about it. “Biology makes things that grow themselves,” she says. “A tree grows itself from sunlight and water, that’s amazing.” At Bioworks2, she hopes to create experimental lifeforms that will really blow college students’ minds. 

ORIGINAL: IEEE Spectrum

By Eliza Strickland

10 Oct 2016

Can we synthetically engineer C4 photosynthesis?

“Photosynthesis as the engine for life on earth has high engineering potential, which has not yet been fully exploited…By step-wise identification of all the components needed for engineering, it will eventually become possible to employ this powerful machinery to increase yields for the future.”

Schuler, ML, Mantegazza, O & Weber, APM, 2016, ‘Engineering C4 photosynthesis into C3 chassis in the synthetic biology age’. The Plant Journal, vol. 87, pp. 62

These lines from the conclusion of the review we write about here are indicative of why so much effort is being put into understanding the more productive C4 photosynthetic system and working to increase important crop yields with it.

Schuler, Mantegazza and Weber’s article in the special issue of The Plant Journal on plant synthetic biology provides an excellent overview of the current status, significant hurdles and possible solutions to those problems of the current research aimed at bolstering rice yield by converting it from the common C3 photosynthesis system to the more efficient C4 system. We’ve previously written about C4 photosynthesis here and here.

C4 photosynthesis

C4 photosynthesis has evolved independently at least 66 times and is likely linked to a sudden drop in atmospheric CO2 levels sometime in the past. It is characterised by the concentration of CO2 around Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), the carbon-assimilating enzyme, reducing the competition that CO2 has with O2 to interact with the enzyme. More CO2 means greater growth and reduced photorespiration, an energy requiring process that is used to remove the O2 reaction products.

The concentration of CO2 in C4 photosynthesis is usually caused by a two-celled (but one-celled is possible) distribution of the process of fixing carbon and the process of reducing it. The two-celled system combines mesophyll (M) cells, which take up the CO2 from the leaf air space, and the bundle sheath (BS) cells, where the Rubisco enzymes reside, the final destination of CO2 for fixation and entry into the Calvin-Benson cycle. These two cells are arranged in concentric layers (called ‘Kranz Anatomy’) around leaf veins, maximising the contact between the two types of cells and increasing the transport of the molecules between them.

M cells convert CO2 to bicarbonate and then into the 4 carbon compound oxaloacetate via an enzyme that doesn’t react with oxygen. The modified compound is then passed to the BS cells where it is reformed into CO2 and fixed by Rubisco to enter the Calvin-Benson cycle.

Basically, by assimilating CO2 away from Rubisco, the plant reduces the ability of Rubisco to interact with O2 and instead it is steadily fed with CO2 from the M cells.

Of course, this description of the process is simplified and although most of the process and main enzymes that carry out the process are known, there are still gaps in our knowledge.

Recent Advances

The gathering of increasing amounts of genomic, trascriptomic and metabolimic data continue to improve our knowledge of C4 photosynthesis, how it evolved and how we might transition C3 crops to use the more efficient carbon fixation method.

Important C4 crop species have had their genomes sequenced and quantitative analysis of transcriptomes have begun to unravel the mystery behind the genes upregulated and downregulated, and the stage of development that these regulatory differences occur, that lead the formation of the Kranz anatomy. What we are finding is that many of the genes involved in C4 photosynthesis exist in C3 plants but are differently regulated at early stages to differentiate the BS and M cells, enable high throughput of metabolites between the cells and to increase the size of vascular tissue to support the increased activity.

Engineering C4 photosynthesis

Our initial attempts to engineer C4 photosynthesis relied on over-expressing one or more enzymes in C3 plants. However, given the enzymes involved in the C4 system are used in the C3 system in multiple alternative pathways, the effects of over-expression were multiple, varied and didn’t have the desire result. The compartmentalisation of reactions, whether in the single or two-celled reactions that make up the distinctive photosystem, is complex.

The notion of being able to engineer C4 photosynthesis is comforted by a number of factors:

1. The main enzymes are already present in C3 photosynthesis;
2. Characteristics such as the passing of metabolites between cells is seen in C3 species such as tobacco plants; and
3. Nature has done it herself in the past on multiple, independent occasions.

But the authors of the paper also note a number of engineering steps that need to be accomplished if we are re-enact evolution ourselves;

1. Higher order veins need to be initiated in plants (it previously being shown that such physical properties were already evolved in plants that subsequently evolved the Kranz anatomy);
2. The ratio of BS to M cells must be increased, ideally in a similar concentric organisation to Kranz anatomy;
3. Enlarging and enriching BS cells with additional chloroplasts;
4. Increasing the connection between M and BS cells;
5. Engineering the different morphologies of the chloroplasts to mimic the morphologies of chloroplasts found in M and BS cells;
6. Mirror the differing roles that M and BS cells take on in C4 photosynthesis so Rubisco reduction of CO2 occurs only in the BS cells with M cells feeding CO2 to the BS cells and excluding the oxidation of O2.

The tools we need

If we are to achieve success we still have some tools to develop and refine.

Chief among this list is a model plant that can be engineered and tested easily with speedy regeneration without requiring too much growing room. The authors point out that rice crops have some limitations in these criteria but identify Brachypodium distachyon as a model C3 plant with a small, annotated genome with quick flowering time, low growing space requirements and an efficient transformation protocol. A model such as this could hasten the engineering, testing and data gathering on conversion which can then be tested on important crop species.

A C4 model plant with similar characteristics is also required. Setaria viridis has previously been suggested as a possible model plant, as has the Fast Flowering Mini Maize.

The ability to drive and control expression of a transgene is also required. Cis-regulatory modules that promote gene expression are still under development in the wider plant synthetic biology area. This leaves a chasm between the tools we have to hand and the possibility that a large number of genes need to be differentially expressed in order to convert C3 photosynthesis to C4 photosynthesis.

Huge strides are being made with genetic manipulation, particularly with the discovery and modification of the CRISPR/Cas 9 system. But, according to the article, the maximum number of genes successfully introduced into a plant, at present, is 9. To induce C4 photosynthesis in a C3 plant, we may need the ability to stably transform a far larger number of genes plus regulatory elements, and do so without disrupting the remainder of the genome or the phenotype characteristics of our food crops.

Even when we do have these tools at the ready, we are still missing some vital information about the genes and regulatory elements that compose C4 photosynthesis. Increasing our knowledge of minutia of genetic composition and regulation of C4 systems compared to C3 systems is still a top priority. Identifying genera with the underlying predisposition that have allowed species within it to evolve from C3 to C4 for comparative analysis, particularly species displaying characteristics of a C3-C4 intermediate with sister taxa displaying C3 and C4 phenotypes, would be idyllic in assisting the study of the evolution. The authors highlight Morandia and Parthenium generas as possible true intermediates between C3 and C4 plants. Programs such as the Grass Phylogeny Working Group and the 1KP (1000 plants) project will greatly assist identifying and genotyping suitable candidates for understanding the genetics behind enhancing crop photosynthesis.

And some suggested means of pushing the research…

It is great to see that not only have the authors elucidated quite extensively the current knowledge and gaps within the field of C4 photosynthesis engineering, but have also suggested a couple of ways of advancing the research.

The first idea they suggested is synthetically replicating a simplified C4 photosynthetic system using known genetic components. The system replicates the targeting of specific enzymes to create a two-celled photosynthesis construct, limiting Rubisco to the BS cells using RNAi to interfere with its transcription in M cells. The article highlights specific transporters that can be used to transport the metabolites between the two cells.

A second suggested idea is using brute force to direct a speedy evolution of a C3 or C3-C4 intermediate species into a C4 plant. Identifying the minimum genetic requirements of a C4 plant in a candidate crop would then be followed by the repetitive growth under the selective pressure of a low CO2 atmosphere. By repeating genomic and transcription analysis of the evolving plant (if successful), a ‘mud-map’ of the road from C3 to C4 plants can be generated and be of enormous use to research seeking to synthetically install the same machinery.

Conclusion

Although its behind a pay-wall, get your hands on this article. Whether it be for a background in C4 photosynthesis or as a springboard for your own research, it is an area of immense potential that should be worthy of an X prize.

ORIGINAL: Legume Laboratory

September 6, 2016

After a secret meeting, scientists announce they are making synthetic human genomes

ktsdesign/Shutterstock.com

It's happening.

An international group of scientists has just announced their plan to create a synthetic human genome within 10 years - which means they're going to try to write a brand new DNA code for human life from scratch.

The ambitious undertaking, called Human Genome Project-write, could be the key to understanding human disease better than ever before, and it could also greatly reduce the cost of genetic sequencing. It's an incredibly exciting project for science, but what's worrying some is the fact that the project has been launched without the public having been properly consulted on any ethical concerns.

Rumours about the new project started last month, when 150 scientists met in a closed-door meeting at Harvard Medical School to talk about building an entirely synthetic human genome.

The fact that journalists weren't allowed to be at the meeting was met with criticism, and now 25 of the researchers have outlined their proposal in Science- although it hasn't done much to relieve concerns. 

Posed as an unofficial follow-up to the hugely important Human Genome Project (HGP) - which ended in 2004 and resulted in the complete mapping of our genetic code - the goal of HGP-write is to take things one step further and not just read our genomes, but create them.

The expectation is that this research, if nothing else, will drop the price of genetic engineering and testing 1,000-fold over the next decade - which would be pretty incredible, seeing as we're already able to sequence an entire genome for under US$1,000 today.

"[T]he goal of HGP-write is to reduce the costs of engineering and testing large genomes, including a human genome, in cell lines, more than 1,000-fold within 10 years, while developing new technologies and an ethical framework for genome-scale engineering as well as transformative medical applications," the researchers wrote in a draft of a press release obtained by

The Washington Post (no official press release has been put out as yet).

To pull this off, the scientists say they'll attempt to raise US$100 million of private and public funding over the next decade, and collaborate with international groups in order to get it done.

And as cool as that would be, they've definitely got their work cut out for them. Although scientists have managed to create synthetic genomes for bacteria the past, writing a complete human DNA code is going to be A LOT harder.

As Bec Crew reported for us back in May, creating a synthetic human genome "means figuring out which chemicals are needed to create the 3 billion bases of DNA that sit inside the 23 pairs of chromosomes found inside every cell nucleus in our body".

Oh, and then they're going to have to work out where all those chemicals go, put them together in lab in the right order, and then arrange them so that they can direct a cell to stay alive.

The good news is that this crazily ambitious project could teach us a whole lot about our biology and disease. But, as someone on Facebook is bound to point out to you today, it could also help scientists get one step closer to creating 'designer babies'.

The concern is that this kind of research could teach us more about how to engineer humans that are resistant to disease, or are exceptionally strong or intelligent. While it's actually not as simple as programming whatever traits we want, it's definitely something we'd be closer to after this project. 

To be very clear, that isn't anywhere near the intention of this project. The researchers state outright that their project will end in the petri dish, and they have no intention of keeping any of the human genome cell lines alive.

But critics are saying that the problem is that the proposal laid out in Science still really doesn't deal with the ethical concerns that it brings up.

The team does write that they "will enable broad public discourse on HGP-write; having such conversations well in advance of project implementation will guide emerging capabilities in science and contribute to societal decision-making", though they don't really outline exactly what questions those discussions will involve. 

There are existing stem cell research guidelines that will apply to their research, but because this is such a new undertaking, the researchers will have the responsibility of creating many new rules as they go.

"Before launching into such a momentous project, questions need to be asked," including whether it should even occur, Stanford University bioengineer Drew Endy told MIT Technology Review. "The authors fail to pose these essential questions. In fact, in their proposal, they fail to pose any questions."

But for all those ethical concerns, the undeniable truth is that this project is probably going to benefit all of us, and our children, in ways we can't even imagine.

"This is as bold an aim as the original human genome project and the authors of this Science paper acknowledge that their new aim will be met with similar controversy as the original HGP had to contend with," synthetic biologist John Ward, from University College London, told the Genetic Expert News Service via email.

"But its now well accepted that the original HGP opened up the possibility and increasingly, the reality, for new medical treatments in human genetic diseases and cancer and we will be reaping the benefits of this for decades to come," he added.

Talking about such an ambitious program again should be incredibly exciting, but as much as we love to see science advance our understanding of biology to all new heights, projects like this need to come with the appropriate level of ethical discussion - if only for the fact that without upfront, transparent discussion, the public is never going to trust what's going on.

And in a world of misinformation, anti-vaxxers, and climate change denial, the last thing we need is to give people a reason to be wary of science.

Let's do this, but let's do it right.

ORIGINAL: Science Alert
FIONA MACDONALD
3 JUN 2016

A programming language for living cells

MIT biological engineers have devised a programming language that can be used to give new functions to E. coli bacteria.
Image: Janet Iwasa

New language lets researchers design novel biological circuits.

MIT biological engineers have created a programming language that allows them to rapidly design complex, DNA-encoded circuits that give new functions to living cells.

Using this language, anyone can write a program for the function they want, such as detecting and responding to certain environmental conditions. They can then generate a DNA sequence that will achieve it.

“It is literally a programming language for bacteria,” says Christopher Voigt, an MIT professor of biological engineering. “You use a text-based language, just like you’re programming a computer. Then you take that text and you compile it and it turns it into a DNA sequence that you put into the cell, and the circuit runs inside the cell.”

Voigt and colleagues at Boston University and the National Institute of Standards and Technology have used this language, which they describe in the April 1 issue of Science, to build circuits that can detect up to three inputs and respond in different ways. Future applications for this kind of programming include designing bacterial cells that can produce a cancer drug when they detect a tumor, or creating yeast cells that can halt their own fermentation process if too many toxic byproducts build up.

The researchers plan to make the user design interface available on the Web.

No experience needed

Over the past 15 years, biologists and engineers have designed many genetic parts, such as sensors, memory switches, and biological clocks, that can be combined to modify existing cell functions and add new ones.

However, designing each circuit is a laborious process that requires great expertise and often a lot of trial and error. “You have to have this really intimate knowledge of how those pieces are going to work and how they’re going to come together,” Voigt says.

Users of the new programming language, however, need no special knowledge of genetic engineering.

“You could be completely naive as to how any of it works. That’s what’s really different about this,” Voigt says. “You could be a student in high school and go onto the Web-based server and type out the program you want, and it spits back the DNA sequence.”

The language is based on Verilog, which is commonly used to program computer chips. To create a version of the language that would work for cells, the researchers designed computing elements such as logic gates and sensors that can be encoded in a bacterial cell’s DNA. The sensors can detect different compounds, such as oxygen or glucose, as well as light, temperature, acidity, and other environmental conditions. Users can also add their own sensors. “It’s very customizable,” Voigt says.

The biggest challenge, he says, was designing the 14 logic gates used in the circuits so that they wouldn’t interfere with each other once placed in the complex environment of a living cell.

In the current version of the programming language, these genetic parts are optimized for E. coli, but the researchers are working on expanding the language for other strains of bacteria, including Bacteroides, commonly found in the human gut, and Pseudomonas, which often lives in plant roots, as well as the yeast Saccharomyces cerevisiae. This would allow users to write a single program and then compile it for different organisms to get the right DNA sequence for each one.

Biological circuits

Using this language, the researchers programmed 60 circuits with different functions, and 45 of them worked correctly the first time they were tested. Many of the circuits were designed to measure one or more environmental conditions, such as oxygen level or glucose concentration, and respond accordingly. Another circuit was designed to rank three different inputs and then respond based on the priority of each one.

One of the new circuits is the largest biological circuit ever built, containing seven logic gates and about 12,000 base pairs of DNA.

Another advantage of this technique is its speed. Until now, “it would take years to build these types of circuits. Now you just hit the button and immediately get a DNA sequence to test,” Voigt says.

His team plans to work on several different applications using this approach: bacteria that can be swallowed to aid in digestion of lactose; bacteria that can live on plant roots and produce insecticide if they sense the plant is under attack; and yeast that can be engineered to shut off when they are producing too many toxic byproducts in a fermentation reactor.

The lead author of the Science paper is MIT graduate student Alec Nielsen. Other authors are former MIT postdoc Bryan Der, MIT postdoc Jonghyeon Shin, Boston University graduate student Prashant Vaidyanathan, Boston University associate professor Douglas Densmore, and National Institute of Standards and Technology researchers Vanya Paralanov, Elizabeth Strychalski, and David Ross.

ORIGINAL: MIT

Anne Trafton | MIT News Office 

March 31, 2016