How to create synthetic DNA

Guido Putignano
Yealthy
Published in
18 min readSep 15, 2022

What is Synthetic Biology:

Synthetic biology is a new interdisciplinary area that involves the application of engineering principles to biology.

It aims at the (re-)design and fabrication of biological components and systems that do not already exist in the natural world. Synthetic biology combines the chemical synthesis of DNA with growing knowledge of genomics to enable researchers to quickly manufacture cataloged DNA sequences and assemble them into new genomes.

Synthetic biologists are keen to develop:

Standardized biological parts:

Identify and catalogue standardized genomic parts that can be used (synthesized quickly) to build novel biological systems.

Applied protein design:

Redesigning existing biological parts and expanding the set of natural protein functions for new processes.

Natural product synthesis:

Engineer microbes to produce all of the necessary enzymes and biological functions to perform complex multistep production of natural products; and

Synthetic genomics:

Design and construct a ‘simple’ genome for a natural bacterium.

Improvements in the speed and cost of DNA synthesis are enabling scientists to design and synthesize modified bacterial chromosomes that can be used in the production of advanced biofuels, bio-products, renewable chemicals, bio-based specialty chemicals (pharmaceutical intermediates, fine chemicals, food ingredients), and in the health care sector as well.

Synthetic biology is creating DNA out of thin air. Synthetic biology and consumer goods describe DNA synthesis as a process where “DNA is created on computers and inserted into organisms. Computers are pretty cool and really useful in synthetic biology labs, but it takes a lot more than a computer to turn a text file full of A’s T’s C’s, and G’s into DNA.

How synthesis of genes has done synthetically:

Most synthetic biologists are not designing gene sequences this way; the field was founded on the idea that we shouldn’t have to build new genetic engineering projects from scratch at all. Instead, synthetic biologists want to build new connections between genes from the library of existing genes that have been sequenced and characterized by other biologists and engineers. Synthetic biologists mix and match genes from different organisms, or alter parts of genes to change how the proteins are expressed or how they function in a living cell. This mix and match of DNA sequences can be built using the “traditional” genetic engineering tools of cutting and pasting DNA using enzymes or outsourced to a synthesis company that will use chemistry to create the DNA molecule.

If you wish to make a DNA molecule from scratch, you must first create some atoms. DNA is an organic chemical molecule made from atoms of carbon, hydrogen, nitrogen, oxygen, and phosphorous. Like many other organic molecules that are made inside living cells, DNA can also be synthesized in test tubes using the tools of organic chemistry. In most descriptions of DNA synthesis technology, we hear that DNA sequences can be made by simply adding together the A’s T’s C’s, and G’s — the “bases” that make the rungs of the twisted DNA ladder. Automated chemical synthesis of DNA begins with DNA bases that have been modified chemically to protect the highly reactive parts of the molecule from binding to each other and creating unwanted side products. These bases and their protecting groups are each made up of a combination of other molecules, each with its own series of chemical reactions, feedstocks, and supply chain economics. For example, the “base” part of adenine (“A”) and guanine (“G”) is a purine ring, which is chemically synthesized by heating formamide at 160–200 degrees Celsius. Formamide is produced through the reaction of carbon monoxide and ammonia. Ammonia is produced by heating nitrogen from the air to high temperatures under high pressure and mixing it with hydrogen, which is produced by burning natural gas, which is extracted from underground reserves by cracking rocks with high-pressure liquids.

In synthetic biology, the physical reality of DNA as a chemical is analogous to the transistors that make up computer chips, its raw sequence of bases the “assembly code.” These are layers that most programmers don’t have to think about when they design software, just like most synthetic biologists don’t necessarily think about how the DNA is made when they design metabolic pathways. But this abstraction in the engineering hierarchy doesn’t mean that the lower levels aren’t important or happen somehow on their own, and certainly not “from scratch.”

It has been said that the 20th century was the “century of the atom” in which discoveries on the physical and chemical properties of the elements led to breakthroughs such as atomic energy (and weaponry), medical diagnostics, computers, and the microchip to name just a few. These advances had a dramatic effect on our way of life and helped shape the promise and possibilities of science and technology. In the early part of the 21st century, we are witnessing what could very likely become known as the “century of DNA.” As the score to life’s intricate symphony, DNA provides the blueprint for biological function. Advances over the last few decades in both reading (sequencing) and writing (synthesis) DNA sequences have made marked changes in our ability to understand and engineer biological systems. These advancements have led to the development of groundbreaking technologies for the design, assembly, and manipulation of DNA-encoded genes, materials, circuits, and metabolic pathways, which are allowing for ever greater manipulation of biological systems and even entire organisms. Thanks to next-generation sequencing (NGS) technologies capable of generating an estimated 15 petabases of sequence data per year worldwide the current metagenomics era has led to the swelling of biological sequence repositories with DNA sequences isolated from every organism and environment imaginable. Associated improvements in bioinformatics techniques and software allow researchers to obtain, analyze, and manipulate these DNA sequences in ways easier than ever. The ever-increasing availability of biological sequence information from all branches of the “tree of life” has deepened our understanding of biological systems and the interrelated nature of organisms at the genetic level. NGS technologies have led to a deeper understanding of human diseases, the microbiome, and the genetic diversity of organisms in our environment. This sequence boom is also allowing for the expansion of scientific disciplines such as metabolic engineering and synthetic biology as researchers seek to use novel sequences in the manipulation of biological systems for anthropocentric means. In addition, this wealth of information is leading to the development of a variety of diagnostics and therapeutics, which will contribute to the long-term improvement of human health.

This biological sequence data bonanza has been aided by a stream of innovations in instrumentation and techniques for generating sequencing data with high fidelity, increased throughput, and decreased cost, which has contributed to making NGS a go-to technology for many applications in biology.

The ability to generate large DNA sequence data sets has allowed researchers to create and test large scoped biological hypotheses using NGS technologies that were not possible before their development. Beyond analyzing biological systems at the DNA sequence level, the need to construct DNA from designed sequences has driven the parallel development of methods and instrumentation to produce synthetic DNA at scale to enable the testing of engineered biological components. DNA reading by NGS when combined with modern DNA synthesis technologies form the two foundational technologies driving synthetic biology efforts and will eventually instill the predictability and reliability to engineered biological systems that chemical engineering has brought to chemical systems. This being said, our ability to sequence DNA is currently better than our ability to synthesize DNA de novo, although new technologies are helping to close the gap.

Synthetic biology is emerging as an important discipline with the potential to impact a number of academic and industrial applications including the creation of novel therapeutics, materials, bio-sensing, and manufacturing capabilities. Although our current understanding of biological systems is vast, it is still far from complete. Adapting exogenous DNA sequences and the functional components they encode that have naturally evolved within a network of interrelated sequences remains a principle challenge of engineering biology. Currently, the engineering of biological systems requires a heavy dose of empirical trial and error to evaluate novel enzymes, expression systems, and pathways for the desired function. Typically, this process is accomplished by first designing a desired synthetic biological circuit or pathway using computer-aided design tools. Next, the resulting construct DNA is divided into smaller overlapping pieces (typically 200–1500-bp segments) that are easier to synthesize. These DNA components are then synthesized from a set of overlapping single-stranded oligonucleotides in-house (or by commercial vendors).

The resulting overlapping synthons are then assembled into larger pieces of DNA and cloned into an expression vector and the sequence of the resulting construct is then verified. The sequence-verified constructs are then transformed into a cell and assayed for function. Depending on the results, changes to the construction design can be made and further iterations of the test cycle repeated. This design, build, test, learn, and repeat process has become the backbone of synthetic biology, which in turn has put a premium on automated processes and methods that can shorten the development cycle and increase throughput. One of the attributes that make biological systems attractive from an engineering perspective is the fact that biological functions are encoded to a large part in DNA. Therefore, a gross simplification of biological engineering can be reduced to the design, production, and testing of DNA sequences. As researchers seek to engineer biological systems with novel DNA sequences, the need for custom synthetic DNA sequences has grown. This is particularly true when the sequences to be engineered are derived from metagenomic sequences in which no organism may be available from which to isolate the DNA via other methods. The synthesis of synthetic DNA is often referred to generically as “gene synthesis,” which specifically is the synthesis of gene-length pieces of DNA (250–2000 bp) directly from single-stranded synthetic DNA oligonucleotides. Unfortunately, whereas the cost of sequencing has decreased precipitously over time, the cost of gene synthesis and oligonucleotide synthesis, in general, has not kept pace, although technological innovation and market forces are progressively lowering the cost of synthetic DNA. The cost of gene synthesis is typically directly tied to the cost of oligonucleotide synthesis from which the genes are made. The cost of oligonucleotide synthesis has not decreased appreciably in more than a decade, generally ranging from $0.05 to $0.17 per base depending on the synthesis scale, the length of the oligonucleotide, and the supplier. Traditionally, this cost floor has been carried through to the production of gene synthesis products. These synthetic genes currently range in cost from $0.10 to $0.30 per bp ($100–$300 for a 1-kb gene) from traditional commercial suppliers, although companies exploiting newer lower-cost synthesis methods are starting to bring the cost down. Lowering the cost of gene synthesis would enable the generation of larger data sets by making the cost of gene construction less expensive, meaning that more constructs could be generated for the same cost investment. This would allow researchers the ability to sample a greater amount of the design landscape. With a greater understanding of what does and does not work in a given construct design, a set of general design rules could be created that would improve the success of the design process and eventually shorten the design-build–test–learn process. Because the major cost of gene synthesis is the reagents that are needed for oligonucleotide synthesis, approaches that reduce reagent consumption, improve the robustness and accuracy of the gene assembly process, and enable increased throughput have become valuable tools for advancing the usage of synthetic DNA by allowing for lower cost of use. In this perspective, a brief overview of the methods and technologies that have contributed to the production of synthetic DNA via gene synthesis methods will be highlighted as well as some of the challenges that have had to be overcome to reduce the cost, increase the throughput, and ensure the fidelity of synthetic DNA.

SYNTHESIS OF LONGER DNA ASSEMBLIES:

Recent advancements in oligonucleotide synthesis and techniques for gene synthesis have largely focused on reducing costs, increasing throughput, and the quality of synthetic DNA. Recent innovations in the design of unique overlapping sequences to direct the assembly process have further expanded the usage of the Gibson assembly method for combinatorial assembly of large DNA sequences.

One of the hallmarks of synthetic biology is the application of rational design principles to the design and assembly of biological components; however, because it is often difficult to know how well a given DNA construct will work once introduced into a cell, it is often necessary to try several versions of the construct to find which one will work best. Therefore, a greater emphasis on the modular design of DNA parts enables the assembly of a greater variety of potential constructs through the mix-and-match combinatorial assembly of DNA components. In addition to simplifying the overall assembly process, modular design and assembly of DNA components make automation of the process possible, which can reduce the time, labor, and cost of making and testing multiple constructs. Most of the aforementioned assembly methods can be used for the assembly in vitro, and Gibson assembly has been applied to the assembly of DNA segments multiple kilobases in length. In another such example, Gibson assembly was used to assemble the 16.3-kb mouse mitochondrial genome directly from 60 mer oligonucleotides. Efficient assembly of even larger synthetic DNA segments can also be performed in vivo by using the homologous recombination capabilities of the yeast Saccharomyces cerevisiae. In an example of the exceptional ability of yeast to assemble exogenous DNA into larger assemblies from overlapping synthons or subassemblies, researchers at the J. Craig Venter Institute have successfully used yeast to assemble multiple 0.5–1 Mbp bacterial genomes and even assembled synthons directly from overlapping oligonucleotides. Each of the aforementioned assembly techniques could be automated to further increase the throughput for constructing larger synthetic DNAs and enable the exploration and testing of large biological hypotheses.

APPLICATIONS FOR SYNTHETIC BIOLOGY?

Synthetic Bio has been used in many applications, and future applications are only limited by human imagination. In industry, Synthetic Bio applications include the production and manufacturing of enzymes, sustainable production of biofuels, and creation of bio-based specialty products. Synthetic biologists are applying engineering principles to biological discoveries to create microbial biosensors for pollutants and to develop microbes or plants for bioremediation of contamination or water pollution in the environment.

In healthcare, synthetic biology is used for rational drug design, immunotherapy for cancers, and creation of medical treatments using sustainable practices. New treatments, such as Car-T therapy for lymphoma and other blood cancers, use synthetic Bio techniques to modify a patient’s immune system so it eradicates their own unique cancer. This breakthrough therapy may potentially replace traditional chemotherapy for cancers such as acute lymphoblastic leukemia.

Agricultural biotechnology (Agbiotech) has and will continue to benefit from synthetic Bioresearch. Applications of Synthetic Bio principles facilitate sustainable farming practices, improve animal health, improve disease resistance and yield of crops, and develop new specialty foods that will reduce our dependence on traditional crops. One of the most important applications for synthetic Bio brings the technology and scientific discovery full circle. As scientists use engineering principles to design biologically based products in agriculture, industry, healthcare, and environmental studies, these above-mentioned explanations extend our knowledge of biological principles. Each new product or solution extends our knowledge of how living things function.

Cost of Synthetic DNA:

The human genetic code has about 3 billion pairs of these letters. The first effort to sequence, or “read” all of these letters took more than a decade and cost billions of dollars. These days, however, anybody’s genetic code can be read for about $1,000. The technology needed to “write” DNA is now undergoing a similar transformation. Over the last decade, the cost of synthesizing a pair of DNA letters has dropped from about one dollar to less than 10 cents.

How Synthetic gene therapy differs:

Efficacy:

Synthetic gene therapy relies on the use of the artificial C3P3 expression technology, which generates large amounts of target mRNA(s). This therapeutic approach therefore radically differs from the standard non-viral gene therapy, which has limited efficacy due to the poor mRNA amounts.

Safety:

The synthetic gene therapy approach has been designed to prevent insertional mutagenesis of the artificial double-stranded DNA in the host-cell genome. Such genetic recombination leads to major safety issues due to the risk of developing solid or hematologic cancers. Synthetic gene therapy therefore radically differs from viral gene therapy, whose uses is limited to ultra-severe diseases.

Tolerance and sustainability:

Artificial DNA used for synthetic gene therapy is entirely devoid of non-methylated CpG dinucleotides, an important driver of the innate immune response that may be accompanied by poor therapeutic tolerance and a loss of efficacy in case of repeated administration.

No size limitations:

Any gene can be expressed by synthetic gene therapy, with no limitation on length of genes.

Some examples related to Synthetic Genomics:

Advancements in genetic engineering and chemical synthesis of genes and whole genomes (‘synthetic genomics’) drive cutting-edge research and novel solutions for practical applications in medicine, energy production, agriculture, and other areas. The most cited examples in which an organism’s entire genome has been synthesized are the construction of poliovirus, the bacteria virus phiX, and the genome for a Mycoplasma bacterium completely from synthetic pieces of DNA. The first two of these examples resulted in the creation of infectious viruses “from scratch,” without the necessity of starting with samples of those viruses. While advancements in genetic engineering and synthesis could help strengthen our response to infectious disease outbreaks, they could also be misapplied to evade a security regime that limits physical access to dangerous pathogens. The ability to synthesize genomes also raises concerns about the degree to which novel organisms might be created that had unpredictable properties.

Biopharma

The oldest application of biotechnology is arguably the biomanufacturing of insulin. Scientists discovered how to co-opt yeast and bacteria into producing human medicines decades ago. But while producing insulin only requires a single gene, there are many important medicines such as Taxol (an anti-cancer drug) that require entire pathways. That’s where synthetic biology comes in.

There is a multitude of companies using synthetic biology to engineer pathways that enable microorganisms to produce medically relevant drugs. Famously, Amyris engineered yeast to produce the antimalarial drug Artemenisin. Synthetic biology can also improve in-vitro drug manufacturing. For instance, Codexis uses synthetic biology to develop more efficient enzymes for the synthesis of small molecule drugs. Beyond the biomanufacturing of drugs, there are many other ways to apply synthetic biology to biopharma research. For instance, there are several companies engineering microbes not only to produce medicines but to deliver them in vivo. These so-called engineered probiotics could potentially be tuned to produce drugs in response to a particular stimulus or only in certain parts of the body. Companies are also engineering human cells for therapeutic purposes. This is the basis for CAR-T cell therapy, a promising new approach to cancer treatment. Beyond single cells, some companies are focused on developing synthetic tissues and whole synthetic organoids for research or therapeutic purposes.

CAR-T cell therapy,

Carbon recycling:

The major goal of the synthetic biology industry is to develop alternative, biology-based methods for industries that typically use petroleum-based products as inputs and produce carbon emissions as outputs. There are many companies working to produce biofuels or bioplastics. For instance, Synthetic Genomics is engineering algae as bio-factories for renewable fuel, and Global Bio-energies is developing processes to ferment plant waste into petrochemical precursors.

Others are working to fix carbon more directly by attempting to optimize natural carbon-fixers (plants and cyanobacteria). Long-term carbon storage is also a challenge, and it’s one some synthetic biologists think bacteria can solve by converting carbon dioxide into a liquid state. Carbon emissions do not only come from burning fuel, however. There are also biological and environmental sources of greenhouse gas. LanzaTech sees these sources as a useful starting point for making high-value chemicals. Its carbon recycling technology platform captures and converts so-called biogas from agricultural and municipal waste, then converts it to biofuels and other products. Together, these efforts comprise what could make up a circular economy in which biology is both the source and the byproduct of many of the products that we depend on instead of petrochemicals.

Fashion and fabric:

We don’t often think about the science behind our clothing, but if you look at the tags of anything you’re wearing right now, you’ll probably be reminded that fashion is fed by a complex mashup of materials. Clothes are a mixture of plant-based materials (like cotton), petroleum-based materials (like nylon and spandex), and animal-based materials (like leather and silk). The fibers that make up our garments are also almost always bleached or dyed and chemically treated. With all this complexity, fashion can have a bit of a nasty environmental footprint.

There are several companies using synthetic biology to come up with greener alternatives for fashion must-haves. For instance, Tinctorium, PILI, and Colorifix are finding a way to dye blue jeans without producing hazardous waste. In addition, Mango Materials are using bacteria to turn methane into bioplastics for clothing and other goods that will degrade naturally if they end up in our oceans as so much waste does. Even fashion icons are taking notice. High fashion designer Stella McCartney is bringing synthetic biology to the runway by partnering with Bolt Threads, a synthetic biology company endeavoring to make synthetic silk and faux leather from mushrooms. Bolt is not alone; synthetic silk companies are popping up all over the world, including AMSilk in Germany and Spiber in Japan, and there’s a company in New York called Ecovative Design that’s using mushrooms to create all sorts of materials for clothing, footwear, and beyond.

Threads by Stella McCartney

Cosmetics and fragrances:

Your clothes may not be the only thing you’re wearing that will soon be shaped by synthetic biology. There are also synthetic biology companies targeting makeup, skin creams, cologne, and perfume. Traditional ingredients for cosmetics are often animal-based, raising purity and animal rights concerns. For example, collagen is a popular ingredient in high-end anti-wrinkle creams, because it’s responsible for skin elasticity. But collagen is sourced from animals, meaning it’s not vegan-friendly, and it can elicit purity and allergy issues. Geltor is using synthetic biology to produce animal-free collagen substitutes. Biossance, an Amyris spinoff, has also used synthetic biology to create an animal-free cosmetic additive, squalane, which was traditionally harvested from shark livers. In addition to taking an environmental toll, ingredients for cosmetics and, especially fragrances, can be incredibly expensive. The essence of grapefruit is captured by a flavor compound called nootkatone. At about $200 per gram, true, pure nootkatone is limited to higher-end colognes. A synthetic biology company called Evolva wants to change that by producing nootkatone and other fragrance compounds via fermentation. Conagen is also using microbial fermentation to produce high-value flavors and fragrances such as vanillin. Boston-based Ginkgo Bioworks produces fragrances using yeast, and has worked with perfumier Robertet and others to develop fragrances and make them at a commercial scale. The company also successfully created a perfume using the floral scents from several extinct flowers.

Food and food ingredients:

This category deserves several posts all its own. Synthetic biology companies are reimagining the food space in several ways from revolutionizing agriculture to tackling food waste to coming up with more environmentally friendly sources of food additives. Starting with the plants in the ground, companies like Pivot Bio and Joyn Bio are engineering soil bacteria to end our dependence on synthetic fertilizers. Other synthetic biology companies are focused on what happens to food after it’s harvested. For example, Conagen is engineering strains of microorganisms and novel enzymes to synthesize all sorts of food additives from food colorings, to sweeteners, to meat tenderizers, to preservatives. Ginkgo spin-off Motif FoodWorks is also using fermentation to brew proteins and nutrients for more delicious and sustainable foods. But companies aren’t just focused on solid ground. AquaBounty, for example, is combining advances in aquaculture with modern genetics to create the world’s most sustainable salmon, while Air Protein is using bacterial fermentation to make protein from the elements comprising the air we breathe. Several companies, such as Memphis Meats and Meatable, are allowing us to have our animals and eat them too, using synthetic biology to create real meat without harming animals or the planet.

The future of synthetic biology

Over the past 30 years, technology fields have seen drastic progress and, to see the same revolution for life sciences, future tools need to allow scientists at the benchtop to iterate the design-build-test cycle just as quickly. Chen highlights that the time it currently takes synthetic biologists to receive DNA is not amenable to progress: “It’s like telling a computer programmer, at Amazon or Apple, that they need to wait up to six weeks to write a particular piece of computer code — it would slow things down to a grinding halt.” DNA sequencing processes becoming quicker and more accessible, “The future of synthetic biology belongs to the people who can provide multi-application experience and the fastest results or products.” The immense potential for an all-in-one integrated system, which both deliver DNA and engineer the desired proteins: “Having these systems at scientist’s fingertips is going to be hands down one of the biggest trends that revolutionize the synthetic biology sector”.

Conclusion

While synthetic biologists offer varying definitions of the field, most will agree that an overarching goal of the industry is to shift our reliance away from chemistry and fossil fuels and toward biology. It’s a lofty goal, but with so many diverse companies working to support it, synthetic biology will have a major role in shaping the future of technology across industries.

References:

[1] https://archive.bio.org/articles/synthetic-biology-explained.

[2] https://blogs.scientificamerican.com/oscillator/genes-cannot-be-made-from-scratch.

[3] https://cshperspectives.cshlp.org/content/9/1/a023812.full.

[4] https://eu.idtdna.com/pages/applications/synthetic-biology.

[5] https://www.npr.org/sections/health-shots/2019/09/24/762834987/as-made-to-order-dna-gets-cheaper-keeping-it-out-of-the-wrong-hands-gets-harder

[6] https://eukarys.com/en/synthetic-gene-therapy.

[7] https://www.aaas.org/resources/minimizing-risks-synthetic-dna.

[8] https://synbiobeta.com/synthetic-biology-is-shaking-up-these-5-industries-some-of-them-might-surprise-you.

[9] https://www.selectscience.net/selectscience-interviews/the-future-of-synthetic-biology-print-dna-at-your-benchtop/?artID=50500

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Guido Putignano
Yealthy
Editor for

Synthetic Biology + Quantum Computing for drug discovery