‘Life at the Speed of Light: From the Double Helix to the Dawn of Digital Life’ by J. Craig Venter (Viking Adult; October 17, 2013)
Table of Contents:
i. Introduction/Synopsis
PART I: THE DAWN OF MODERN GENOMICS: FROM THE DISCOVERY OF THE DOUBLE HELIX TO THE HUMAN GENOME PROJECT
1. The Discovery of the Structure of DNA: The Double Helix
2. How Genes Translate into Physiological Features: Protein Coding
3. Reading DNA: The Dawn of Genetic Sequencing
4. Sequencing Human Genes
5. Sequencing Whole Genomes of Living Organisms
6. Mycoplasma Genitalium and the Smallest Possible Genome
7. The Human Genome Project
8. The Dawn of Genetic Engineering: Gene Splicing
PART II: SYNTHESIZING LIFE
9. The Dawn of DNA Synthesis
10. Synthesizing the First Full Genome: Synthetic Phi X 174
11. Creating the First Synthetic Genome of a Living Organism Part I: The E. coli Approach
12. Creating the First Synthetic Genome of a Living Organism Part II: The Brewer’s Yeast Approach
13. Creating the First Synthetic Life Form
a. Transplanting a Natural Genome into a Foreign Species
b. Transplanting a Synthetic Genome into a Foreign Species Part I: Synthetic Mycoplasma Genitalium into Mycoplasma Pneumonia
c. Transplanting a Synthetic Genome into a Foreign Species Part II: Synthetic Mycoplasma Mycoides into Mycoplasma Capricolum
PART III: THE PRACTICAL (AND NOT SO PRACTICAL) APPLICATIONS OF SYNTHETIC BIOLOGY AND SYNTHETIC GENOMICS
14. What Is Life?
15. The Practical Applications of Genetic Manipulation
16. Synthetic Genomics and Influenza Vaccines
17. Synthetic Genomics and Improved Antibiotics
18. Synthetic Genomics and the Search for Alien Life
19. Conclusion
i. Introduction/Synopsis
Ever since the structure of DNA was deciphered by James Watson and Francis Crick in 1953, the field of biology has advanced at a lightning-quick pace. In this time, we have learned how DNA codes for the manufacture of proteins of which every living thing is made, and thus acts as the blueprint of life. We have also learned to read this blueprint; to splice it (to transfer genes, and hence features, from one organism to another—and even one species to another); to synthesize it from its component parts; and we have even learned to rewrite DNA to yield wholly new biological products, features and organisms. Thus recent advances have not only allowed us to gain a better understanding of what life is and how it works, but have also allowed us to take control of life and to manipulate it to help advance our ends—and in fields as wide-ranging as food production, medicine, energy, environmental protection etc. And this is just the beginning, for biologists still have much to learn about which genes code for what features, and how to manipulate DNA to achieve the best results—and thus we can expect that some of the greatest applications to come out of biology are yet to come.
The biologist J. Craig Venter has been at the forefront of biological research for the past 35 years, and has played a pivotal role in some of its most important advances (including everything from sequencing the human genome, to creating the first synthetic life form), and in his new book Life at the Speed of Light: From the Double Helix to the Dawn of Digital Life, Venter takes us through the major advances that have occurred since the time of Watson and Crick—and also touches on what is likely to come next.
After taking us through the basics of DNA, Venter touches on the advances that led up to his effort to sequence the entire 3-billion-letter human genome. This story includes all of the major advances in biologists’ ability to read DNA, and culminates with the success of the human genome project.
From here we are taken through biologists’ efforts to move from reading DNA to synthesizing it in the lab. Once again, Venter and his collaborators have played a central role in these advances, including being responsible for the latest and greatest accomplishment here—which involved synthesizing a modified version of the genome of a single-celled organism, booting it up inside a recipient cell, and having it survive, thrive and reproduce. Venter gives a detailed account of this accomplishment, and thus we are given an inside view into the scientific process—with all its trials, tribulations, and glorious successes.
Finally, Venter details where biology is headed now, and next—including where his own research is taking him. Here we learn about the cutting-edge of synthetic biology, which is the attempt to transform biology into an engineering science. Specifically, we learn how biologists are continuing to perfect the art of manipulating DNA, and how this is leading to exciting new applications across many fields. To give just one example, take Venter’s work with influenza vaccines. Venter is in the process of using synthetic biology to design, manufacture, and deliver influenza vaccines in a fraction of the time that it now takes—work that promises to save millions of lives in the event of future influenza outbreaks.
On the more speculative side of things, Venter ventures into how new advances might be used to probe for life in other parts of the universe—and how the genomes of any such life might be read, and sent back to earth on the back of electromagnetic waves to be synthesized and recreated in the lab. Life at the speed of light indeed!
Here is J. Craig Venter introducing his new book:
*To check out the book at Amazon.com, or purchase it, please click here: Life at the Speed of Light: From the Double Helix to the Dawn of Digital Life. The book is also available as an audio file from Audible.com here: Audio Book
The following is a full executive summary of Life at the Speed of Light: From the Double Helix to the Dawn of Digital Life by J. Craig Venter.
PART I: THE DAWN OF MODERN GENOMICS: FROM THE DISCOVERY OF THE DOUBLE HELIX TO THE HUMAN GENOME PROJECT
1. The Discovery of the Structure of DNA: The Double Helix
The age of modern genomics truly begins with the discovery of the structure of DNA in 1953. This discovery had been sought by numerous ambitious biologists, but it was James Watson and Francis Crick who would ultimately solve the puzzle—work for which they would later (in 1962) receive the Nobel Prize (loc. 513).
At the time, it was known that DNA is made up of “building blocks called nucleotides, consisting of a deoxyribose sugar, a phosphate group, and four nitrogen bases—adenine (A), thymine (T), guanine (G), and cytosine (C)” (loc. 501), and that the nucleotide bases always came in pairs (loc. 504); however, it was not known just how these building blocks joined up with one another to form the structure that is DNA.
Drawing on the X-ray photography of DNA of Maurice Wilkins, Rosalind Franklin and Raymond Gosling, Watson and Crick were able to surmise how the building blocks pieced together into a two-stranded spiral, or double helix. As Venter explains, “it was Wilkins who had shown Watson the best of Franklin’s X-ray photographs of DNA. The photo numbered fifty-one… taken by Raymond Gosling in may 1952, revealed a black cross of reflections and would prove the key to unlocking the molecular structure of DNA, revealing it to be a double helix, where the letters of the DNA code corresponded to the rungs. On April 25, 1953, Watson and Crick’s article ‘Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid’ was published in Nature. The helical DNA came as an epiphany, ‘far more beautiful than we ever anticipated,’ explained Watson, because the complementary nature of the letters—component nucleotides—of DNA (the letter A always pairs with T, and C with G) instantly revealed how genes were copied when cells divide” (loc. 513).
Below is pictured the X-ray image that Watson and Crick used to decipher the structure of DNA, and below that are Watson and Crick revealing the structure of DNA itself:
As the quote above makes clear, the reason why the discovery of the structure of DNA was so pivotal was because it immediately laid bare how DNA replicates itself—and thus how genes are copied in cell division and passed down from one generation to the next. Specifically, it now became clear that individual genes were to be found in the precise sequence of nucleotide bases, and that this sequence can be easily recreated by way of splitting the double-stranded structure down the middle, and having free-floating nucleotides join up with each of the strands, creating two new double-stranded structures (as pictured below).
*The story of the race to discover the structure of DNA is a truly fascinating one, and is told brilliantly in the following 1 hour documentary: The Secret of Life Documentary
2. How Genes Translate into Physiological Features: Protein Coding
Still, what was not yet known was just how the information contained in each gene was translated into physiological features. In other words, it was not known how genotypes translate into phenotypes. It did not take long for biologists to discover this, though.
Indeed, by the end of the 1960s the process by which genes code for the manufacture of proteins (of which all living tissue is composed) had been unlocked (loc. 110). Here is Venter with the short version of how this works: “The DNA software is ‘transcribed,’ or copied, into the form of a messenger RNA (mRNA) molecule. In the cytoplasm, the mRNA code is ‘translated’ into proteins” (loc. 534). And here is Venter with the longer version of how this works: “the most important breakthrough in molecular biology, outside of the genetic code, was in determining the details of the master robot—the ribosome—that carries out protein synthesis and so directs the manufacture of all other cellular robots. Molecular biologists have known for decades that the ribosome is at the focus of the choreography of protein manufacture. To function, the ribosome needs two things: a messenger RNA (mRNA) molecule, which has copied the instructions for making a protein from the storehouse of DNA genetic information in the cell; and transfer RNA (tRNA), which carries on its back the amino acids used to make the protein. The ribosome reads the mRNA sequence, one codon at a time [a codon is a chain of 3 nucleotide bases], and matches it to the anti-codon on each tRNA, lining up their cargo amino acids in the proper order. The ribosome also acts as a catalyst, a ribozyme, and fuses the amino acids with a covalent chemical bond to add to the growing protein chain. Synthesis is terminated when the RNA sequence codes for a ‘stop,’ and the polymer of amino acids must then fold into its required structure to be a biologically active protein” (loc. 648).
The following is an excellent animation of this process—as the video indicates, there is an updated version of the animation, but I personally think the draft is better:
3. Reading DNA: The Dawn of Genetic Sequencing
The next step for biologists was to learn how to read the sequence of nucleotide bases in a strand of DNA, so that they might begin the work of discerning which sequences code for what proteins and physiological features (or phenotypes).
This they did, but at first the work of reading (or sequencing) DNA was painfully slow. As Venter explains, “in the 1960s and 1970s, progress was slow, and sequencing was measured in terms of a few base pairs per month or even year. For example, in 1973 Allan Maxam and Walter Gilbert, of Harvard University, published a paper describing how twenty-four base pairs had been determined with their new sequencing method. Meanwhile, RNA sequencing was also underway and progressed a bit faster. Still, compared with the abilities of today’s technology, the effort required to read even a few letters of code was heroic” (loc. 805).
In the mid-1970s, though, the biologist Fred Sanger developed a couple of new methods for sequencing DNA that represented a big step forward. As the author explains, “the DNA-sequencing technology that made it possible for me to sequence the human genome originated in the mid-1970s, when Fred Sanger’s team in Cambridge developed new DNA-sequencing techniques, the first being ‘plus-minus’ sequencing, followed by what Sanger named dideoxy DNA sequencing but which in his honor is now called Sanger sequencing. Sanger sequencing uses dideoxynucleotides or terminator nucleotides to stop the DNA polymerase from adding nucleotides to the growing DNA chain; dideoxynucleotides lack a hydroxyl (-OH) group, which means that, after being linked by a DNA polymerase to a growing nucleotide chain, no further nucleotides can be added. By attaching a radioactive phosphate to one of the four nucleotides, to label the fragments, it was possible to read the order of the As, Ts, Cs, and Gs by exposing the gel used to separate one base from another to X-ray film” (loc. 821).
In 1977 Sanger was able to use his new sequencing technique to read the first full genome—that of the phi X 174 bacteriophage virus, which consists of 5,386 bases (loc. 826). This was a significant accomplishment. Still, while Sanger sequencing represented a major step forward, it remained somewhat painstaking and awkward; and, as Sanger himself concluded, “‘[It] seemed that to be able to sequence genetic material a new approach was desirable’” (loc. 834).
This new approach would come in the mid 1980s, when the biologist Leroy Hood discovered a way to automate (and hence radically speed-up and increase the accuracy of) Sanger sequencing. As the author explains, “Leroy Hood and his team at Caltech published a key paper describing how they replaced the radioactive phosphate with four different fluorescent dyes on the terminator bases of DNA, which, when activated with a laser beam, could be read sequentially into a computer” (loc. 839).
With this the era of automated genetic sequencing had finally begun.
4. Sequencing Human Genes
It is at this point that Venter himself personally enters the story. In 1984, Venter had moved to the National Institutes of Health (NIH), and subsequently began work sequencing human genes (loc. 834). With the technology of the day, this was exceedingly slow and laborious work. As the author explains, “during my first year at NIH we sequenced only one gene, the human-brain adrenaline receptor, using the radioactive Sanger sequencing, but it took the better part of a year” (loc. 834).
Progress sped up significantly, though, with the introduction of the new automated technology. Venter was one of the first biologists to get his hands on a new automated sequencer, and he and his team used it to quickly sequence thousands of human genes (loc. 844).
This work was aided by a short-cut that Venter had developed to pick out just the expressed portions of a genome—the genes themselves. (Not all of the nucleotide sequences in a genome are used to code for protein manufacture. Other nucleotide sequences serve different functions, such as switching other genes on and off, and some sequences have no discernible function at all.) Specifically, Venter had taken the tack of identifying genes in messenger RNA (in the form of what his team called expressed sequence tags [ESTs]), converting this RNA back to DNA, and then sequencing the DNA. As the author explains, “using the new DNA-sequencing technology coupled with computer analysis, my lab rapidly sequenced thousands of human genes by a new method I had developed which focused on relatively short sequences which my team had named expressed sequence tags (ESTs). The EST method involved sequencing the expressed genetic material, messenger RNA (after converting it into complementary DNA)” (loc. 845).
The quick sequencing of large numbers of human genes was certainly a major achievement. However, the accomplishment would soon be overshadowed by controversy, as the US government made a move to file patents on the genes that were sequenced—a move that was met with deep opposition (and was later abandoned) (loc. 845).
5. Sequencing Whole Genomes of Living Organisms
In any event, by the early 1990s Venter had received an attractive offer to set up his own research institute stocked with the latest in automated sequencing technology (loc. 850). The author leapt at the opportunity (and named his new institute The Institute for Genomic Research [TIGR]), and it is here where he began the hard work of sequencing whole genomes of living organisms.
By this point, Venter had teamed up with the eminent biologist and Nobel laureate Hamiltion (Ham) Smith, and the two began work sequencing the genome of the first living organism (as mentioned above, Fred Sanger had already succeeded in sequencing a virus—the phi X 174—but viruses are not technically living organisms, as they must parasitize a foreign genome in order to reproduce) (loc. 1045). The genome that Venter and Smith decided to sequence first was that of the Haemophilus influenza bacterium (loc. 859).
Even using the latest automated technology of the day, reading this genome was going to be a tall order, as it consists of over 1.8 million base-pairs (loc. 872). In order to tackle the problem, Venter’s team decided to employ an indirect (though time-saving) strategy. Specifically, rather than reading the base-pairs in order, 1 by 1, the team would blast apart the genome into fragments, read the fragments, and then decipher the order of fragments to determine the proper sequence (a technique that would be dubbed ‘whole genome shotgun sequencing’ [loc. 888]). As Venter explains, “unlike Sanger’s lab years earlier with phi X 174, which used isolated unique restriction fragments for sequencing one at a time, we relied completely on randomness. We broke up the genome into fragments in a mixed library and randomly selected twenty-five thousand fragments to obtain sequence reads of around five hundred letters each. Using a new algorithm developed by Granger Sutton, we began to solve the greatest biological puzzle to date, reassembling those pieces into the original genome. In the process we developed a number of new methods to finish the genome. Every single one of the base pairs of the genome was accurately sequenced and the twenty-five thousand fragments accurately assembled. The result was that the 1.8 million base pairs of the genome were re-created in the computer in the correct order” (loc. 872).
Being the first to successfully sequence the genome of a living organism was certainly a major achievement. But the team didn’t stop there. They went on to identify all of the genes in the genome, and to explore how these genes accounted for how the bacterium works—including how it causes meningitis and other infections (loc. 872-80). At the same time, the team also began work sequencing the genome of a second living organism—that of Mycoplasma genitalium (loc. 884).
While Venter and his team were busy working away, rumors began to spread about the remarkable achievements that were happening in the lab, and thus Venter was invited to speak at the 1995 annual meeting of the American Society of Microbiology, in Washington D.C. (loc. 877).
It was at this event that Venter (with Smith alongside) made the team’s achievements public. In his speech, Venter highlighted the following: “with Haemophilus influenza we had transformed the double helix of biology into the digital world of the computer, but the fun was only now beginning. While we had used its genome to explore the biology of this bacterium and how it causes meningitis and other infections, we had in fact sequenced a second genome to validate the method: the smallest one known, that of Mycoplasma genitalium. When I ended my speech, the audience rose in unison and gave me a long and sincere ovation. I had never before seen so big and spontaneous a reaction at a scientific meeting. That was a very sweet moment. My team had become the first ever to sequence the genetic code of a living organism, and of equal significance was the fact that we had done so by developing a new method, which we named ‘whole genome shotgun sequencing.’ This feat marked the start of a new era, when the DNA of living things could be routinely read so that they could be analyzed, compared, and understood” (loc. 888).
6. Mycoplasma Genitalium and the Smallest Possible Genome
It was mentioned above how Venter and his team had already proceeded to sequence the genome of a second living organism—that of Mycoplasma genitalium. As explained in the previous paragraph, the main motivation behind this move was to provide a way to double-check and validate the new gene sequencing technique that the team had innovated. Now we must understand why it was this particular species that Venter decided to sequence next.
It was mentioned in the previous section that the genome of M. genitalium is the smallest one known. In fact, it consists of just 582,970 base-pairs (and 524 genes) (loc. 1400, 909). This made it such that it could be sequenced very quickly. Indeed, Venter’s team needed just 3 months to do so (loc. 896). This was certainly an important consideration in choosing it. However, there was another reason behind sequencing M. genitalium that was even more important: At the time, Venter had become interested in exploring the question of what is the minimum genome that is needed to sustain life; and M. genitalium—with its already miniscule genome—provided the perfect opportunity to do just this (loc. 913-17).
Thus following the achievement of having sequenced the genomes of the first life-forms, Venter and his team eventually (after sequencing several more full genomes [loc. 927-44, 953, 966-71]) got to work on the smallest-genome-needed-for-life question—using M. genitalium.
Having sequenced M. genitalium, the next step in using it to explore the minimum genome question was to experiment with knocking out each of its 524 genes, 1 by 1, in order to determine which ones are vital for life and which are not. As Venter explains, “the most obvious approach was to knock out genes in the M. genitalium genome to try to establish which are essential: remove or disable a gene and, if the organism continues to live, you can assume that particular gene did not have a critical role; if the organism dies, the gene was clearly essential” (loc. 974).
When Venter’s team undertook these experiments, though, they discovered that the question of which genes are necessary for life is somewhat thornier than expected, because different environmental conditions can render different genes vital. As the author explains, “once we had completed our analysis of the data, we realized that this absolute scoring system was naive and that genes and genomes are context-specific… Because all cells obtain key nutrients and chemicals from their environment, if the environment changes, then the genes that are required for life in that new environment also need to change” (loc. 999).
Now, there is a way around the environmental problem; however, it requires having full knowledge of what each gene is and does (loc. 1006), and at this point Venter and his team still lacked full understanding of 152 genes (32%) of M. genitalium (loc. 920). Given that this was the case, Venter’s team could only estimate the number of genes in M. genitalium that are absolutely essential. As the author explains, “from our combined studies we estimated that in M. genitalium 180 to 215 genes are nonessential and that the number of essential genes is 265 to 350. Of the latter, 111 [were] of unknown function. This was clearly not the precise definition of life that we were seeking. In addition, working through this data, it became increasingly obvious that the genes that were individually dispensable might not be able to all be deleted together” (loc. 1020).
The team was at a cross-roads; however, there was a way out. It’s just that this way out would require a mammoth and unprecedented effort. As Venter explains, “given the limits of the molecular-biology tools… we concluded that the only way to get to a minimal genome would be to attempt to synthesize the entire bacterial genome from scratch. We would have to chemically synthesize the entire chromosome, using only the essential genes” (loc. 1020).
Now, chemically recreating, or synthesizing DNA had in fact been achieved by this point; however, the longest stretches of DNA that had been successfully synthesized were but a few thousand base-pairs long (loc. 1031). Synthesizing the genome of a living organism would require the team to create a string of DNA some 600,000 base-pairs in length, and would require whole new techniques and methods (loc. 1031). Thus the team was well aware that they had their work cut out for them. Still, Venter was eager to get the project underway.
Just as Venter’s team began the project, though, an opportunity arose that Venter simply could not turn down: the opportunity to sequence the entire 3-billion-base-pair human genome.
7. The Human Genome Project
Surprisingly, Venter has very little to say about the Human Genome Project in the book (he mentions it in passing several times, but never delves into any detail). We can only speculate that this is because the author feels he has said enough about this project elsewhere, and/or because once automated sequencing (and the shotgun method) had been invented, there was little additional new science that went into the feat. Also—and this is pure speculation—but one gets the impression that Venter was happy to get the human genome project out of the way, so that he could get on to the much more challenging—and important—task of synthesizing life. (Nevertheless, the race to sequence the human genome is a very interesting story, and it is told well in the documentary here: The Human Race Documentary.)
In any event, before we move on to the story of how Venter successfully synthesized life, we must first mention one other major achievement in biology that had occurred while the author was busy reading human genes and sequencing life. And that achievement is gene splicing—which is what originally made genetic engineering possible.
8. The Dawn of Genetic Engineering: Gene Splicing
Gene splicing involves removing a gene from one organism and inserting it into another organism, so that that second organism expresses the transferred gene. The feat was first achieved in the early 1970s. As Venter explains, “the 1970s brought the beginning of the gene-splicing revolution, a development potentially as revolutionary as the birth of agriculture in the Neolithic Era. When DNA from one organism is artificially introduced into the genome of another and then replicated and used by that other organism, it is known as recombinant DNA. The invention of this technology was largely the work of Paul Berg, Herbert Boyer, and Stanley Norman Cohen. Working at Stanford, Berg began to wonder whether it would be possible to insert foreign genes into a virus, thereby creating a ‘vector’ that could be used to carry genes into new cells. His landmark 1971 experiment involved splicing a segment of the DNA of a bacterial virus, known as lambda, into the DNA of a monkey virus, SV40. Berg would share the 1980 Nobel Prize in Chemistry for his work, but he did not take the next step of introducing recombinant DNA into animals. The first transgenic mammal was created in 1974 by Rudolf Jaenisch and Beatrice Mintz, who inserted foreign DNA into mouse embryos” (loc. 563).
Gene splicing was the premise behind virtually all of the major innovations and applications in the first wave of genetic engineering (which lasted from the early 1970s to the late 1990s). These innovations included the use of recombinant DNA to produce human insulin (which, in 1982, was the first biotechnology product to reach the market) (loc. 584), as well as all of the genetically modified crops that made headlines in the 1980s and 1990s.
Starting in the early 2000s, though, biologists began developing several more ways to manipulate DNA, and were able to gain far more control over the blueprint of life—thus opening up a whole new world of applications. The new techniques and methods represent such a departure from the first generation of genetic manipulation that biologists have now begun referring to the practice by a separate name: synthetic biology.
As the moniker suggests, synthesizing biological parts and products (including DNA) is a big part of the practice; and, once again, Venter and his collaborators played a big part in developing the sophisticated from of DNA synthesis that is now commonly used. Much of this work was done in the past decade, beginning in the early 2000s, which is when Venter turned his attention from the human genome project to the enterprise of synthesizing the first genome, and then synthesizing the first organism—which is the topic of Part II.
PART II: SYNTHESIZING LIFE
9. The Dawn of DNA Synthesis
The practice of chemically recreating, or synthesizing, DNA was first accomplished in the 1950s—not long after the first successful attempts at reading it (loc. 1025). However, as with reading, synthesizing would not be automated until many years later, in the 1980s. As Venter explains, “work on the chemical synthesis of DNA dates back to the 1950s, with the success of Har Gobind Khorana and Marshall Nirenberg, but it was only in the 1980s that substantial progress was made, following the invention of the automated DNA synthesizer by Marvin Caruthers, at the University of Colorado, Boulder. His synthesizer uses four bottles containing the DNA bases A, T, C, and G, and adds one base to another in a prescribed order. In this way, DNA synthesizers can make short stretches of DNA called oligonucleotides” (loc. 1025).
One issue with Caruthers’ synthesizer, though, is that it is subject to the occasional error (when a designated base fails to latch-on to the chain [loc. 1158-62]), and thus the longer the stretch of DNA you are synthesizing, the more likely there is to be an error in the sequence (loc. 1025). This is a significant problem because, as we shall see, the smallest error in a DNA sequence can mean the difference between life and non-life (loc. 1177). Also, as mentioned in the previous paragraph, Caruthers’ synthesizer is limited in how long a DNA chain it is capable of creating. Given these limitations, whenever you are trying to synthesize a string of DNA of any considerable length, the only viable way to proceed is to break it into fragments, synthesize the fragments, check the fragments to ensure they are accurate, and reassemble the fragments to form a whole.
As mentioned above, when Venter and his team began the project to synthesize the genome of the first living organism, the longest chain of DNA that had been successfully synthesized consisted of only a few thousand base-pairs—and the process of linking-up very long fragments of DNA had yet to be achieved. Thus the team recognized that they were entering uncharted territory, and would have to develop new methods and techniques. As the author explains, “when we first started to discuss synthesizing an entire genome, the largest pieces of DNA that had been made measured only a few thousand base pairs. To build the genome of a viable organism required us to chemically synthesize and assemble almost six hundred thousand base pairs, and as a result we knew that we would need to develop new methods to accomplish this goal” (loc. 1032).
10. Synthesizing the First Full Genome: Synthetic Phi X 174
To test the viability of the ultimate goal, the team decided to begin with a more modest task—that of synthesizing a virus (and specifically the 5,384 base-pair phi X 174 virus—the first virus to have been read) (loc. 1032). Unfortunately, the team’s first attempt to synthesize phi X 174 was a failure, due to an overabundance of errors in the synthesizing process (actually, this early failure turned out to be a harbinger of how difficult the ultimate task would be); however, the team came back with a second attempt, and this time scored success.
Here’s how the procedure worked: the team broke down the 5,384 base pairs of the genome into 259 oligonucleotide fragments of 42 base pairs each (such that each of the 259 fragments had some room to overlap with its neighbors) (loc. 1181). They then ‘purified’ each of the oligonucleotides in order to weed out some of the possible errors (loc. 1189). Following this, the team mixed the oligonucleotides together, and added some chemical glue (the enzyme DNA ligase) (loc. 1193). The glue succeeded in binding together many (though not all) of the fragments, such that the team now had “assemblies with average sizes of around seven hundred bases” (loc. 1197).
In order to bind the remaining fragments together the team used a process known as polymerase cycling assembly (PCA). In this process, the double-stranded fragments of DNA are split in two and left to connect-up with the overlapping portions of the opposite strand of their rightful neighbors (with the remaining un-complemented bases being coupled with individual nucleotides [and more glue]) (loc. 1197). This process worked to bind the remaining fragments together, and the team now had their full 5,386 base pair genome (loc. 1208).
To make sure the genome was viable, the team tested it to see if it would properly infect an E. coli bacteria. And it did! As the author explains, “the synthetic bacteriophage DNA had indeed had the ability to infect, reproduce, and then kill the bacterial cells. Ham and Clyde were giddy with excitement. Our entire process to create the synthetic genome and infect the cells had taken us just two weeks” (loc. 1225).
Now that the team had discovered that synthesizing a full viable genome was possible, it was time to go after the grand prize: synthesizing the genome of a living organism: “having accurately made DNA fragments five thousand bases in size, we realized that we had solved a key limitation in DNA synthesis and could take the next step. We were now ready to attempt to go where no one had gone before, to create a whole bacterial synthetic genome and try to produce the first synthetic cell” (loc. 1281).
11. Creating the First Synthetic Genome of a Living Organism Part I: The E. coli Approach
As mentioned above, the species that Venter and his team had wanted to synthesize first was that of Mycoplasma genitalium. As you will recall, the genome of M. genitalium consists of some 582,970 base pairs—a good 100 times larger than that of phi X 174. Unfortunately, synthesizing M. genitalium would not be a simple matter of repeating the steps that went into constructing phi X 174; rather, several whole new processes would need to be invented (loc. 1404).
Since Venter’s team had successfully synthesized phi X 174, they knew that they could construct a chain of DNA in the range of 5000-7000 base pairs in length (loc. 1397). Thus the team decided to approach the M. genitalium genome by way of splitting it into fragments, or ‘cassettes,’ of roughly this size, and then trying to patch these cassettes together: “for the genome synthesis we divided the M. genitalium genome into one hundred and one segments that we called ‘cassettes,’ each around the size of the phi X 174 genome… our plan was to find a way to combine them to reconstruct the M. genitalium genome (loc. 1398).
Also, the team decided to modify the original genome somewhat (by adding a string of nucleotide bases to it), to prove that the final product was indeed synthetic, and not merely a copy of the original biological genome: “Rather like artists sign their work, we wanted to leave a signature in the new genome to distinguish it from a natural genome. So, using single amino-acid code abbreviations, we designed ‘watermark’ sequences that would spell out ‘Venter Institute’ and ‘Synthetic Genomics,’ as well as the names of the key scientists working on the project” (loc. 1448).
When it came to patching together the cassettes of the genome, the first patching technique that Venter’s team experimented with involved exploiting the natural DNA patching ability of a species of bacteria known as Deinococcus radiodurans (loc. 1463). This incredible bacteria is able to patch-up and repair its genome even after being exposed to over 3 million rads of ionizing radiation (comparatively, just 500 rads is capable of killing a human being), and thus Venter felt that the team might be able to harness this natural ability to help patch together their cassettes (loc. 1480-84). Unfortunately, after much effort and experimentation, the technique proved to be ineffective (loc. 1488).
The second approach that Venter and his team devised was quite a bit more complicated than the first. To knit the cassettes together in the second approach, the team first took each cassette and chemically chewed off some bases at the ends of half of the double-stranded structure (loc. 1496). They then brought neighboring cassettes together and allowed them to connect-up with one another out of the chemical attraction of the free bases (loc. 1500). As the author explains, “to knit the pieces together, we added to the DNA mixture in the tube an enzyme (known as a 3’-exonuclease) that chews the end of the DNA, digesting only one of the DNA’s two strands… and leaving the other strand… exposed. Using changes in temperature to control the exonuclease, we could ensure that the corresponding single-stranded ends of the cassettes would find each other and stick together, due to the chemical attraction of the complementary bases on each strand… To ensure that we ended up with complete double-helix strands, we then added DNA polymerase, as well as some free nucleotides, so that any place where the 3’-exonuclease chewed away too much of the strand, the polymerase would fill in the missing bases” (loc. 1500).
Following this, the team took the newly adjoined cassettes and cloned them by way of introducing them into the genome of an E. coli bacteria, “so that, as this organism multiplied, so did copies of the larger fragment” (loc. 1488). The larger fragments were then removed from the E. coli, and the patching process repeated (loc. 1504-08). This entire process was then to be repeated until all of the cassettes were joined (loc. 1504-12).
Everything was proceeding swimmingly until the second-to-last stage of the assemblage process. By this time the team had 4 fragments of 144,000 base pairs (loc. 1512). But this is where things went sideways. As Venter explains, “as we reached the penultimate step—producing half-genome segments of 290,000 base pairs by combining two of the four quarter-segments together—we ran into problems: the 290kb segments appeared too large for E. coli to accommodate” (loc. 1516).
This was a big disappointment. But all was not lost, for the team thought that they might still be able to salvage their approach if they could find an alternative to E. coli that could accommodate their larger fragments (loc. 1516).
12. Creating the First Synthetic Genome of a Living Organism Part II: The Brewer’s Yeast Approach
The best candidate was a species called Saccharomyces cerevisiae (better known as brewer’s yeast) (loc. 1521). As Venter explains, “for centuries S. cerevisiae has been used for alcohol fermentation as well as for making bread, but in the laboratory it has been routinely exploited because it has a relatively small genome and an array of genetic tools that make genetic manipulation easy. For example, S. cerevisiae uses what is called homologous recombination, in which segments of DNA with sequences on its ends similar or identical to those in the S. cerevisiae genome can be spliced into its genome, replacing the intervening sequence” (loc. 1525).
Fortunately, the brewer’s yeast worked like a charm! As the author explains, “we found that by using the yeast cloning, we could stably grow our large synthetic DNA constructs, and by using the yeast homologous-recombination system, we could link our overlapping one-quarter genome segments to form one-half genome pieces. This system then allowed us to assemble the entire M. genitalium genome in yeast. The end of the long, hard climb to the first synthetic genome of a living organism now seemed to be in sight” (loc. 1533).
The only work that remained now was to remove the synthetic genome from the yeast genome, and read it to ensure that the sequence was in fact correct (loc. 1537-41). Happily, everything went according to plan, and the sequence was a match: “We were all very pleased and relieved when the DNA sequence exactly matched our computer-designed sequence, including the watermarks we had introduced. We had synthesized a 582,970-base-pair M. genitalium genome, and in achieving that feat we had created the largest synthesized chemical molecule with a defined structure” (loc. 1545).
The team named the new synthetic genome M. genitalium JCVI-1.0, and submitted their results to the Journal Science. The results were published online on January 24, 2008, and in print on February 29 (loc. 1545).
13. Creating the First Synthetic Life Form
Half the battle was won, but the second half of the battle yet remained, as the team still needed to take their new genome, transplant it into a cell, and have it function and reproduce like a normal genome. As the author explains, “we celebrated our success in creating the genome but knew that our biggest challenges were yet to come: we now had to find a way to transplant the first synthetic genome into a cell, to see if it could function like a normal chromosome. In the process, the host cell would be transformed into one where all the components were manufactured according to the instructions held in our synthetic DNA” (loc. 1549).
The simplest approach would have been to transplant the synthetic genome into a cell of the same species as the genome itself (M. genitalium). However, this would have left some ambiguity as to whether the transplanted genome was recreating the cell machinery anew, or whether it was borrowing from pre-existing structures (loc. 1809). Thus the team needed to transplant the synthetic genome into the cell of a foreign species.
Now, not only had this never been achieved (obviously), but the more straightforward procedure of transplanting a natural (non-synthetic) genome into a foreign species had never been achieved (loc. 1602-10), and thus the team decided to begin with this more modest task (in order to demonstrate that the more complicated feat was in fact achievable in principle).
a. Transplanting a Natural Genome into a Foreign Species
The two species that the team decided to experiment with were Mycoplama mycoides and Mycoplasma capricolum—with the former being the donor species, and the latter being the recipient (loc. 1646-58) (the reason the team did not choose to experiment with a natural M. genitalium genome is that this species is very slow to reproduce, and thus any experiments involving it take weeks to complete—a fact that would later haunt the team [loc. 1646]).
After many arduous trials, the team was eventually able to successfully transplant an M. mycoides genome into the M. capricolum cell, and have it completely take over the cell. Essentially, the procedure used chemicals to isolate and purify an M. mycoides genome from its home cell (loc. 1675), and then a separate chemical process to inject the genome into the M. capricolum cell so that it could take over the cell (loc. 1698). After investigating the modified M. capricolum cell, the team could be sure that the process had in fact worked. As Venter explains, “these data provided us with the conclusive evidence that the only proteins in the transplanted cell were those resulting from transcription and translation of the transplanted M. mycoides genome. We could now be absolutely confident that we had a new and novel mechanism to transform the genetic identity of a cell that does not involve DNA recombination or natural mechanisms… We now knew that we had the first cells derived from the deliberate transplantation of one species’ genome into a host cell from another species. In doing so, we had effectively changed one species into another” (loc. 1766).
The team now had proof that it was possible to take a genome and transplant it into a foreign species (loc. 1766). Thus the next project would be to attempt to take a synthetic genome and transplant it into a foreign species (loc. 1774).
b. Transplanting a Synthetic Genome into a Foreign Species Part I: Synthetic Mycoplasma Genitalium into Mycoplasma Pneumonia
As we know, the team had already successfully generated a synthetic genome (that of M. genitalium). Thus the team now needed to pick out a recipient species for the transplantation process. The team chose Mycoplasma pneumonia (loc. 1868).
Unfortunately, the team met with difficulties straight away, as it turned out that the process they had used to remove the synthetic M. genitalium genome from the yeast genome left it too damaged to be viable (loc. 1804-08).
In response, the team decided to try and develop a new, more delicate technique to retrieve a foreign genome from a yeast cell. Once again, the team used the genome of M. mycoides to experiment with (loc. 1824). After many trials, the team were finally able to retrieve an M. mycoides genome from a yeast cell in a form that allowed it to be transplanted into an M. capricolum cell (loc. 1820-62).
Unfortunately, though, when the team tried the same approach with the synthetic M. genitalium genome, the experiment failed (loc. 1868). This time, the team discerned, the problem had nothing to do with the donor genome, but with the recipient cell. As Venter explains, “we discovered, eventually, that the recipient M. pneumonia cells contained a nuclease on their cell surfaces that chewed up any DNA to which it was exposed” (loc. 1868).
In order to proceed as planned, the team would now have to come up with a way to skirt the M. pneuomonia nuclease problem. By this time, though, the team—and especially Venter—had become very frustrated with working with M. genitalium (loc. 1869). The reason for this, as mentioned above, is that M. genitalium multiplies at a very slow rate (just once every 12 hours [loc. 1642]), and thus any experiments involving it take upwards of 6 weeks (as opposed to just days with other varieties of bacteria) (loc. 1642, 1812).
However, giving up on M. genitalium at this point would mean having to create a whole new synthetic genome from a different species—and one that would have to be much longer than the relatively miniscule 600,000 base pairs of M. genitalium. Given how difficult it had been to synthesize the latter, one would think that this would be out of the question. However, while Venter and his team had been busy working away at synthesizing a genome, one of the team members, Dan Gibson, had come up with a new technique to synthesize DNA that greatly simplified and sped up the process (loc. 1872).
The new synthesizing technique (called the ‘Gibson assembly’ [loc. 1879]) made the option of switching to a new species at least viable. But still, the team was initially divided on what to do. Venter himself wanted to change course and drop the very frustrating M. genitalium, but much of the rest of the team were reluctant to abandon the genome they had spent so much time and effort creating (loc. 1884-92).
After careful consideration, though, the team finally came around to the idea of starting over with a new synthetic genome (loc. 1892). This time the team would work to synthesize an M. mycoides genome, and attempt to transplant it into an M. capricolum cell (loc. 1888). The rationale here was that the team had already successfully transplanted a natural (non-synthetic) M. mycoides genome into an M. capricolum cell (loc. 1883-88). What’s more, the team had also successfully retrieved a natural M. mycoides genome from a yeast cell (loc. 1883). Thus all of the pieces of the puzzle were in place—it remained only to put them together.
c. Transplanting a Synthetic Genome into a Foreign Species Part II: Synthetic Mycoplasma Mycoides into Mycoplasma Capricolum
Using the Gibson assembly process, the team was able to synthesize the genome of M. mycoides fairly quickly (loc. 1904-112). Thus the way was paved for the team to now attempt to transplant the genome into an M. capricollum cell. On the team’s first attempt, though, the effort failed (loc. 1913-16).
In an attempt to discern why, the team went back and double-checked the sequence of their synthetic genome (loc. 1916). For though the team had in fact sequenced the genome before attempting the transplant, they felt it was possible for an error in the sequencing to have slipped through (loc. 1916).
Sure enough, upon double-checking the sequence the team now found a single error in the M. mycoides genome—and what a big error it was! As Venter explains, “we sequenced the DNA once again, this time using the highly accurate Sanger sequencing method, and found that there was a single base-pair deletion… This is called a ‘frameshift mutation’; in this case, the frameshift occurred in the essential gene dnaA, which promotes the unwinding of DNA at the replication origin so that replication can begin, allowing a new genome to be made. That single base deletion prevented cell division and thus made life impossible” (loc. 1936).
Thankfully, the error was relatively easy to fix, for the team simply had to go in and add the base where it had been missing, and then reassemble the genome (loc. 1939). The necessary corrections were made, and the experiment ran again. This time, finally, success was achieved. As Venter explains, “On April 21 [2010] we had the results from sequencing the DNA from the living synthetic cells, and there was no longer any room for doubt: the cell had been controlled only by the genome that we had designed and synthesized. The sequence showed our genome to have the 1,077,947 base pairs, exactly as intended… as well as the four watermark sequences, a critical proof that the DNA was synthetic. Just as we had suspected, a one-letter deletion out of over one million base pairs of DNA had made the difference between life and no life / We named our new cell M. mycoides JCVI-syn 1.0 and worked on getting our results ready for publication… On the day of its publication online, May 20, 2010 (the print version appeared July 2), media from around the world gathered in Washington, D.C., for our press conference. Joined by editors from Science, we announced the first functioning synthetic genome” (loc. 1985 / 2011).
Here is that announcement:
PART III: THE PRACTICAL (AND NOT SO PRACTICAL) APPLICATIONS OF SYNTHETIC BIOLOGY AND SYNTHETIC GENOMICS
For Venter, the value of his team’s work and accomplishments lies in 2 main areas. The first is that the work greatly adds to our knowledge and understanding of life itself, and the second is that the work opens up numerous broad avenues for practical applications. Let us begin with the understanding aspect first.
14. What Is Life?
One question that has nagged at humanity in our quest for understanding is just what is the basis of life? Living things have a vitality that clearly distinguishes them from mere material objects, and thus many have postulated (or assumed) that the basis of life must be some immaterial force, or power—a spirit, say, or a soul. As the science of biology has progressed, though, this belief (known as vitalism) has come under increasing pressure.
For Venter, the successful synthesis of a life-form does much to answer, once and for all, the question regarding what is the basis of life. And the answer is this: life is an information system—one that is based on the letters of DNA (and thus vitalism has no part to play in the matter). As the author explains: “My thinking about life had crystallized as we conducted this research. DNA was the software of life, and if we changed that software, we changed the species, and thus the hardware of the cell… Our experiments did not leave much room to support the views of the vitalists or of those who want to believe that life depends on something more than a complex composite of chemical reactions. These experiments left no doubt that life is an information system” (loc. 1774). Elsewhere, the author adds that “when my team successfully booted up synthetic DNA software within a cell, we demonstrated that our basic understanding of the machinery of cellular life had advanced to a significant point. In answer to Erwin Shcrodinger’s little question ‘What is Life?’ we had been able to provide one compelling answer: ‘DNA is the software and the basis of all life’” (loc. 2088).
Still, Venter acknowledges that the book on vitalism cannot be closed entirely. The reason for this is that Venter’s team borrowed from a living system in order to re-create life; and unless a wholly novel organism is created entirely from the ground up beginning with non-biological building blocks only, the gap between the biological world and the non-biological one cannot be said to be closed entirely. As the author explains, “because we began with an existing cell and all its protein machinery, the question remains whether modern cells, which are the result of billions of years of evolution, can actually be recreated form the basic components of life. Can we… utilize the isolated protein and chemical components to boot up a synthetic chromosome and in the process create a new kind of self-replicating cell? Can we grow in the laboratory an organism that represents a brand-new branch on the tree of life, a representative of what some like to call the Synthetic Kingdom? In theory, at least, we can. The science of the coming century will be defined by our ability to create synthetic cells and to manipulate life… When we achieve that milestone, we will have opened a new chapter in our understanding of life and, I believe, will have a complete answer to Schrodinger’s difficult question” (loc. 2101).
So, just what evidence is there that biologists may, ultimately, be able to recreate life from scratch? Plenty, in fact. For even now, biologists have shown that several decidedly biological molecules can be created purely out of basic, non-biological elements and compounds—including, most impressively, several building blocks of genetic molecules. As Venter explains, “the nature of chemistry at the dawn of life—prebiotic chemistry—takes us back to 1952 and the famous experiments by Stanley Miller and Harold Urey, at the University of Chicago. Complex organic molecules, including sugar and amino acids, were formed spontaneously from water (H20), ammonia (NH3), hydrogen (h2), and methane (CH4) when the latter were exposed to conditions (a closed, sterile system with heat and sparks from electrodes) that simulated those thought to have been present on the early Earth. A few years later, at the University of Houston, Joan Oro found that the nucleotide base adenine and other RNA and DNA nucleobases could form spontaneously from water, hydrogen cyanide (HCN), and ammonia” (loc. 2118).
In principle, then, yes, creating life from scratch should be an achievable accomplishment; and indeed some biologists are even now working on making this a reality (loc. 2151).
15. The Practical Applications of Genetic Manipulation
While the ability to create life from scratch may yet lay in the future, the ability to read, manipulate, modify, and now synthesize the blueprint of life—DNA—are all already here. As you will know, these abilities have already yielded many important practical applications. Here is Venter to give a brief recap of the accomplishments to this point: “the quest to manipulate life in the laboratory has come a long way since the early days of recombinant DNA, in the 1970s, when Paul Berg, Herbert Boyer, and Stanley Cohen began to cut and splice DNA. By the end of that decade a laboratory strain of E. coli had been genetically altered to produce human insulin. Since then scientists have induced bacteria to manufacture human clotting factors to treat hemophilia and to make growth hormone to treat dwarfism. In agriculture, DNA has been altered to make plants resistant to drought, pests, herbicides, and viruses; to boost their yields and nutritional value; to manufacture plastics; and to reduce the use of fossil-fuel-based fertilizer. Animal genes have been altered in attempts to increase yields, to produce models of human disease, to make drugs such as anticoagulants, and to produce ‘humanized’ milk, and pig organs that can be transplanted into people. Genetically modified cells have been used to manufacture proteins, from antibodies to erythropoietin that increases the production of red blood cells. Some patients have been genetically altered via gene therapy, in which a software ‘patch’ is used to treat genetic disorders such as immune deficiency, blindness, and the inherited blood condition beta-thalassemia” (loc. 1365).
Today, biologists have taken their ability to manipulate and modify DNA, and other cell structures, to extraordinary heights. As the author explains, “genes from almost every species, including bacteria, yeast, plants, and mammals, have been or are being cloned and studied on a daily basis. Metabolic pathways are being engineered in research laboratories and in biotech companies to coax cells into generating products ranging from pharmaceuticals to food and industrial chemicals to energy molecules” (loc. 586).
And now that synthesizing DNA has been added to biologists’ tool-kit, the potential practical applications have only expanded. Venter and his team ran through the possibilities in broad terms even as they announced their successful project back in May of 2010 (as seen in the video above) (loc. 2019). But we shall now take a closer at one or two of the applications that are now underway.
16. Synthetic Genomics and Influenza Vaccines
One of the more significant applications of synthetic genomics currently underway is Venter’s project in the design, production, and delivery of influenza vaccines. In the event of an influenza outbreak, time is of the essence in producing and delivering effective vaccines, and this is where synthetic genomics stands to make a big impact—and in several ways.
To begin with, the development of vaccines has traditionally relied upon the use of fertilized hen eggs, and is a process that takes upwards of 35 days (loc. 2688). With synthetic genomics, however, the hen eggs can be done away with, and the process it