Scientists One Step Closer to Artificial Life and the Best Case for ID
About a year ago my family and I (along with some good friends) rode mules down to the bottom of the Grand Canyon. That night we ate dinner at the Phantom Ranch and the next day rode back out of the canyon. In just two short days, I developed a real admiration for the animals we rode. The ability of those mules to maneuver on rugged and narrow trails overlooking sheer drops was impressive.
Since the time of the Neolithic revolution humans have relied on domesticated animals to meet our needs. These creatures provide us with an ongoing source of food and serve as beasts of burden, transporting humans and materials from place to place and plowing fields.
In the last few decades, life scientists have come to rely on their own beasts of burden in their research endeavors. Creatures like Rhesus monkeys, mice, fruit flies, nematodes, yeast, bacteria like E. coli, and a whole host of others have all played critical roles in the development of modern biology. Without these model laboratory organisms, biologists could never have performed the necessary experiments to understand how living systems work. In fact, E. coli and the yeast Saccharomyces cerevisiae are playing a central role in scientists’ quest to create artificial life in the lab.
As I pointed out a few weeks ago, Craig Venter, one of the pioneers in the field of genomics, recently founded a company called Synthetic Genomics. This group is devoted to creating artificial, nonnatural microbes that have commercial utility, particularly for the production of ethanol, hydrogen, and other forms of renewable energy.
Scientists like Venter who pursue the creation of artificial and synthetic life claim that these novel life-forms will benefit humanity. Still, the very real prospect of scientists creating life in the lab also raises theological concerns. Should human beings “play God?” Additionally, many people believe that if scientists can create life in the lab, it would prove that there is nothing special about life itself.
Venter’s team has recently achieved another milestone in the quest to create an artificial life-form in the lab. Researchers at Synthetic Genomics improved upon the method of synthesizing and cloning (in yeast) the entire genome of a wild-type M. genitalium. Ironically, instead of supporting an evolutionary origin of life, this research empirically demonstrates that life’s origin and transformation cannot happen apart from the work of a mind.
Last week, I gave a description of the strategy the team is taking to synthesize and assemble the entire genome of M. genitalium and explained the first stages of its production. This week, I would like to continue describing the effort needed to synthesize and assemble an organism’s whole genome.
Synthesis of a Genome
The Synthetic Genomics scientists didn’t just rush into the lab and start throwing nucleotides into test tubes and running chemical and enzymatic reactions. Instead, they carefully devised a synthesis strategy. They decided to build the genome by first synthesizing small pieces of DNA and then assembling these pieces into increasingly large sections using chemical, biochemical, and in vivo methods until the entire genome was cobbled together.
Before they began any lab work, the research group started at the drawing board. They carefully parsed the sequence of the entire M. genitalium genome into fragments (called cassettes) each about 5,000 to 7,000 nucleotides (bp) in size. They delineated the boundaries between cassettes so that these demarcations would reside between genes. They also carefully designed the cassettes so that the sequences between two adjacent pieces of DNA overlapped by about 80 bp. This planning allowed them to piece together the M. genitalium genome in a manageable and orderly fashion.
Once the cassette map was developed they executed the synthesis and assembly in stages that included:
- using automated DNA synthesizers that utilize chemical and physical processes to synthesize and purify, respectively, about 10,000 short pieces of the genome approximately 50 bp in length;
- use of enzymes biochemically combine the chemically made fragments into 101 larger fragments (about 5,000 to 7,000 bp each) that corresponded to the cassettes they mapped out at the drawing board stage;
- use of enzymes and the bacterium Escherichia coli to combine the 101 larger fragments into four fragments about 140,000 bp each;
- use of yeast to combine the four fragments into the entire genome.
Each stage of this process demanded careful planning and execution.
Once the chemical synthesis and purification of the 50 bp oligonucleotides has been completed, they need to be linked together to ultimately form the 5,000 to 7,000 bp cassettes diagramed onto the M. genitalium genome map. Chemists don’t have enough control over the reactions that generate the oligonucleotides to efficiently and accurately perform this crucial step. The difficulty can be side-stepped by using enzymes to carry out the recombination process.
Once again, biochemists can’t just toss the DNA fragments into a test tube with a mixture of enzymes and get the desired recombinations. Instead they have to painstakingly formulate a strategy that includes selecting the appropriate enzymes based on their catalytic properties, designing the oligonucleotides–prior to the chemical synthesis step–so that they are compatible with the enzymes, and devising a reaction scheme that will yield the desired recombination product.
Venter’s group had previously worked out the procedure used to recombine the 50 bp oligonucleotides into fragments about 5,000 to 7,000 bp by putting together the entire genome (5,386 bp) of a bacterial virus. The process entails:
- Treating the oligonucleotides with the enzyme T4 polynucleotide kinase. This enzyme modifies the ends of the oligonucleotides so that they can take part in the next stage of the reaction.
- Treating the end-modified oligonucleotides with Taq ligase. This enzyme combines smaller oligonucleotide fragments into larger ones, preparing them for the next stage of the recombination. The oligonucleotides from each of the DNA strands must pair up with each other in the appropriate way. For this to happen, it was critical that Venter’s team carefully design the sequences of the 50 bp oligonucleotides made by chemical synthesis.
- Performing a polymerase cycling assembly of the paired and ligated oligonucleotides. Again, the success of this step depends on the careful design of the sequences of the 50 bp oligonucleotides made by chemical synthesis. This cleverly designed procedure uses enzymes called DNA polymerases to assemble small paired DNA fragments into larger ones. The paired oligonucleotides do not fully overlap with one another. Due to their partial overlap, single stranded regions exist. The DNA polymerases fill in those gaps, eliminating the overlap and in the process joining the fragments together, extending their size. The polymerase cycling assembly has to be repeated between 35 to 70 times to build fragments between 5,000 and 7,000 bp.
- Performing a polymerase chain reaction amplification of the fully assembled DNA pieces, 5,000 and 7,000 bp. This step not only generates numerous copies of the fully assembled DNA pieces, it also eliminates any partially assembled DNA if the design of this step is done in such a way to allow only the fully assembled DNA pieces to be amplified.
Once the 101 5,000-7,000 bp cassettes were put together, they were further combined in three stages to form four pieces of DNA about 144,000 bp each. These large fragments each equaled one fourth of the M. genitalium genome.
In stage one, the researchers assembled neighboring cassettes in groups of four (1-4, 5-9, 10-13, etc.) along with a segment of DNA from the bacterium E. coli to form pieces of the M. genitalium genome about 24,000 bp each. This stage of the assembly yielded 25 24,000 bp fragments.
The specific steps for this stage included:
- Treatment of the 5,000-7,000 bp oligonucleotides with an enzyme called a 3’ exonuclease. This enzyme removes pieces of DNA that are part of the paired oligonucleotides from each of the DNA strands to expose overlapping sequences.
- The oligonucleotides were then allowed to incubate for a period of time under exacting conditions to allow the neighboring cassettes to assemble.
- Treatment of the assembled oligonucleotides with a polymerase and ligase to fill in the missing nucleotides (removed as a result of the exonuclease treatment) and link the assembled cassettes together.
Assembled cassettes were also joined to a piece of DNA from the bacterium E. coli. Each piece of bacterial DNA served as a marker specific to each of the 24,000 bp cassettes. Because of the unique DNA sequences, the bacterial segments allowed researchers to import the assembled DNA into E. coli so that it could be cloned and amplified for the next stage.
After cloning and amplifying the partially assembled genome, the pieces of bacterial DNA were released. The 24,000 bp fragments were ready to be moved on to the next stage of construction.
During stage two, the scientists joined three adjacent 24,000 bp pieces of DNA to form 72,000 bp fragments utilizing the same enzymatic protocol as used in the first stage of the assembly. Stage three involved combining two adjacent 72,000 bp pieces to form 144,000 bp fragments.
At this point in the building process, the researchers discovered that enzymes and E. coli proved useless since the microbe can’t handle larger DNA fragments. To finish the genome assembly the researchers turned to yeast.
Recombination in Yeast
Venter’s team wisely chose the yeast Saccharomyces cerevisiae to complete the construction of the M. genitalium genome because it can take up extremely large pieces of a foreign genome when combined with DNA that is compatible with yeast. Instead of using enzymes in a test tube to complete the genome assembly, they used the yeast’s biochemical machinery inside the cell to put together the final pieces of the genome before they cloned it. Venter and his coworkers found it unnecessary to bring together the genome in a stepwise fashion (first joining together two quarter genomes followed by two half genomes). Instead they could induce the yeast to take up all four pieces of the genome simultaneously to achieve the assembly. The capacity of S. cerevisiae to take up several pieces of DNA and unite them all at once has intrigued the team with the possibility that added efficiencies could be built into their approach to total genome synthesis and assembly.
Recently Venter’s group showed that stages two through four could be eliminated. Instead of relying on enzymatic recombination together with cloning in E. coli to successively produce 72,000 bp and 144,000 bp pieces of DNA, they could use yeast to recombine the twenty-five 24,000 bp DNA fragments generated at stage one of the process.
The complete chemical synthesis and assembly of the entire M. genitalium genome accomplished by the people at Synthetic Genomics represents a tremendous scientific achievement. The editors at Science voted this work as one of the top ten scientific breakthroughs of 2008 because it will pave the way to better understanding of the minimum requirements for life and because it introduces a key technology in the creation of novel, nonnatural life-forms for commercial and biomedical use. It also has important implications for the creation/evolution controversy. I’ll discuss these implications next week.