Posted by Fazale ‘Fuz’ Rana, Ph.D.
Scientists One Step Closer to Artificial Life and the Best Case for ID
Sometimes the solutions to problems emerge from unexpected places. The Human Genome Project holds potential for understanding and treating human disease. Surprisingly, it also may play a role in addressing concerns about climate change and the need for renewable, clean-burning energy.
Even though the Human Genome Project offers the real possibility of transforming health care and medicine, its greatest impact may not be the advances it spurs in medicine and the understanding of human biology. Rather, history is likely to view its greatest achievement as spawning the scientific discipline called genomics.
Genomics, which cross-sects genetics, molecular biology, biochemistry and computer science, focuses on sequencing and characterizing the entire DNA content (called the genome) of organisms. Scientists hope that this endeavor will allow them to gain new insight into the biology and comparative relationships among life (both living and even extinct). Some scientists also think that genomics may be the gateway to creating artificial life in the lab.
As I pointed out last week, 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—thus, the link between the Human Genome Project and the climate and energy crises.
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 raises all sorts of theological questions. Should human beings “play God”? Additionally, many people believe that if scientists can create life in the lab, then there is nothing special about life itself because its origin could have easily taken place on the early Earth without God’s involvement.
Venter’s team has recently achieved another milestone in the quest to create an artificial life-form. Researchers at Synthetic Genomics improved upon the method to synthesize and clone (in yeast) the entire genome of a wild-type M. genitalium. Ironically, instead of providing support for an evolutionary origin of life, this research provides an empirical demonstration that life’s origin and transformation cannot happen apart from the work of a mind.
Last week, I provided an overview of the strategy Venter’s team is taking to generate artificial life from the top down and gave a brief report on their progress to date. This week, I would like to begin the process of detailing the most recent work performed by his team. Along the way, I will describe the effort and ingenuity needed to synthesize a genome starting with the four nucleotides that comprise DNA (the molecule that makes up an organism’s genome). Next week I will continue discussing the effort needed to synthesize and assemble an organism’s whole genome. In two weeks, I will look at the implications of this work for the creation/evolution controversy.
Synthesis of a Genome
To achieve the total synthesis of the entire M. genitalium genome, Venter’s team 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 DNA pieces into increasingly large sections using chemical, biochemical, and in vivo methods until the entire genome was cobbled together.
The Strategy
Before they began any lab work, the scientists started at the drawing board, carefully parsing the sequence of the entire M. genitalium genome into fragments (called cassettes), 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 to 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.
Chemical Synthesis and Purification
The capacity to affect the chemical synthesis of DNA has been a long time in the making. This process refer to the nonbiological, chemical production of small segments of DNA, called oligonucleotides.
With state-of-the art methods, chemical synthesis can reliably generate oligonucleotides up to about 200 bp in size using automated procedures. The first experiments towards this end began in the 1950s and advanced through several key milestones, thanks to the efforts of some of the most prominent chemists of the last half century.
DNA consists of chain-like molecules known as polynucleotides. Two polynucleotide chains align in an antiparallel fashion to form a DNA molecule. The paired polynucleotide chains twist around each other to form the well-known double helix. The cell’s machinery forms the individual polynucleotide chains by linking together four different subunit molecules called nucleotides. The four nucleotides used to build DNA chains are adenosine, guanosine, cytidine and thymidine, denoted as A, G, C and T, respectively.
The individual nucleotides have several different chemical groups that can react with each other to form bonds between two nucleotides. Yet in DNA, the nucleotides must be linked together in a specific way to form the polynucleotide strands. Enzymes that constitute the part of the cell’s machinery that makes DNA have a high degree of specificity, ensuring that the linkages form in the correct manner. In a test tube without enzymes present, the nucleotides will react with each other to form a variety of linkages. This results in a polynucleotide with a mish-mashed structure and no biological use.
To get around this problem, chemists have devised a strategy that relies on the use of chemical groups to block these reactive sites. Each group has been specifically chosen and designed to uniquely fit the individual chemistry of each reactive site. The blocking groups can be selectively removed by chemists at the appropriate times during the synthetic sequence to make specific chemical groups available to react. When the total synthesis of the oligonucleotides has been achieved the remaining blocking groups are then removed.
If these reactions are conducted in solution, a purification step has to be included after each nucleotide addition to the growing oligonucleotide chain. To get around this cumbersome step, chemists have devised an approach using a solid support. The oligonucleotide chain is anchored to the solid support during the reaction sequence. The first nucleotide is attached to a solid material that has been packed into a column. Chemicals can be poured into the column to initiate reactions with the next nucleotide in the chain. When the reaction is complete, the column is washed. Unreacted materials and undesired side products are removed from the column, while the oligonucleotide remains attached. This not only eliminates a costly purification step, it also increases the accuracy of the synthesis.
The specific steps for the chemical synthesis of oligonucleotides include:
- Attaching the first nucleotide to a solid support after deblocking the appropriate group.
- Removing the appropriate protecting group from the attached nucleotide in the first position to allow it to react with the next nucleotide in the oligonucleotide sequence that has had the appropriate blocking group removed from it.
- Allowing the nucleotides to react under carefully controlled conditions.
- Adding a chemical cap to any unreacted oligonucleotides. (This step is necessary because a small percentage of the nucleotides don’t react with the attached oligonucleotide chains.)
- Stabilizing the linkage between the two nucleotides with an oxidizing agent
- Washing away unreacted nucleotides.
- Repeating steps 2 through 6 until the entire oligonucleotide has been made.
- Cleaving the oligonucleotide from the solid support.
- Removing all the blocking groups.
- Purifying the oligonucleotide. This step is absolutely critical to ensure the accuracy of the biochemical recombination steps during the whole genome assembly process.
Because the technology to chemically synthesize oligonucleotides has been in place for several decades, it’s easy for scientists to take it for granted. Today, the chemical production of oligonucleotides is virtually an automated turnkey process. Yet, it’s important not to overlook the remarkable technical accomplishment this capability represents. If it wasn’t for the dedicated efforts over the course of the last half century of some of the best scientists in the world (including Nobel Laureates), to develop and improve methods to chemically make DNA, Venter’s team would have no hope of affecting the complete synthesis of the M. genitalium genome.
Next week I’ll continue to describe the steps needed to synthesize and assemble the entire M. genitalium genome by focusing on the biochemical and in vivo assembly of the chemically-synthesized pieces of DNA into the whole genome.
Thanks for hanging in there. The reward comes in discovering a significantly bolstered case for intelligent design.
As a student I came across the humorous definition of a nuclear physicist as one who was “learning more and more about less and less, until finally he knew everything about nothing.” In today’s world of research, this reference to the wonders of nature at its tiniest levels could also be said about the biologist. Every day we are treated to new discoveries revealing the amazing intricacies of the biological cell and the molecular machines that govern its functionality, all at a size that requires an electron microscope to even begin to see.
Does the universe end at the farthest reaches we can observe? If it doesn’t, then what characteristics does this realm beyond the observable universe exhibit? While science fiction authors have written in depth about alternate universes, many scientists want to bring these questions into their arena. To do so, they must posit explanations that account for existing data and predict what future experiments and observations might reveal. The most expedient avenue for such investigations involves