Today’s New Reason To Believe

Fazale ‘Fuz’ Rana, Ph.D.

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.

The Strategy

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.

Biochemical Recombination

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.

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:

1. Attaching the first nucleotide to a solid support after deblocking the appropriate group.
2. 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.
3. Allowing the nucleotides to react under carefully controlled conditions.
4. 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.)
5. Stabilizing the linkage between the two nucleotides with an oxidizing agent
6. Washing away unreacted nucleotides.
7. Repeating steps 2 through 6 until the entire oligonucleotide has been made.
8. Cleaving the oligonucleotide from the solid support.
9. Removing all the blocking groups.
10. 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.

Posted by Fazale ‘Fuz’ Rana, Ph.D.

Scientists One Step Closer to Artificial Life and the Best Case for ID

Mary Shelley’s famous Gothic horror story presents a fascinating character: Victor Frankenstein. As a burgeoning scientist, Victor becomes obsessed with discovering the “principle” that distinguishes life from inanimate matter. After many long hours of study and laboratory work he uncovers “the cause of generation and life,” and becomes “capable of bestowing animation on lifeless matter.”

Frankenstein exercises his newfound ability by bringing a humanoid monster to life, only to abandon it in disgust. Victor’s moral failings lead to several tragic deaths at the hands of his creature, including those of his brother, his best friend, and, later, his wife on their wedding night. Even though the scientist and the creation are somewhat sympathetic figures, it’s not clear who’s the true monster—Victor, the creature, or both.

If there is a real life Frankenstein, he might very well be Craig Venter.

Venter stands as perhaps one of the most important scientists in the last decade or so. (In 2008, Time magazine voted him one of the world’s 100 most influential people.) As a major player in the emergence of the science of genomics, he is a scientific maverick who thinks big and has little patience for the red tape and bureaucracy that characterize many scientific programs. And like Victor, he is a polarizing figure, much admired and much hated by people within and outside the scientific community.

Recently, Venter founded a company called Synthetic Genomics. As with Shelley’s protagonist, Venter wants to create life in the laboratory. The new company is devoted to creating artificial, nonnatural life microbes that have commercial utility, particularly for the production of ethanol, hydrogen, and other forms of renewable energy. Once again, Venter has generated a mixture of excitement and horror among the scientific community, and the public at large.

Scientists like Venter who pursue the creation of artificial and synthetic life claim that these novel life-forms will benefit humanity. If they accomplish the desired breakthrough, it could go a long way toward resolving the energy and climate crises.

The very real prospect of scientists creating life in the lab raises all sorts of theological questions. Should human beings “play God”? Some conservative Christians worry that the genesis of novel life-forms by human hands eliminates the need for a Creator by substantiating the evolutionary paradigm. Many theists and atheists, alike, believe that if scientists can create life in the lab, then there is nothing special about any life. Therefore, the origin-of-life could have easily taken place on the early Earth without God’s necessary involvement.

The latest work by Venter’s team stands as another milestone in the quest to create an artificial life-form in the lab. Ironically, instead of supporting an evolutionary origin of life, this research demonstrates that life’s beginnings and transformation cannot happen apart from the work of an intelligent agent.

This week I will discuss the progress Venter and his team at Synthetic Genomics have made toward achieving their goal. In the next couple of weeks I’ll describe the details of their most recent accomplishment and will eventually explore the implications of this work for the intelligent design (creation)/evolution controversy.

The Path to Artificial Life

Venter and his coworkers became interested in creating artificial life as an outgrowth of another project. Initially, they were interested in determining the minimum genome for life. The term “genome” refers to an organism’s entire hereditary information, stored in the nucleotide sequences of DNA. The information housed in genomes exists in units called genes. These units contain the information that the cell’s machinery uses to make proteins.

Proteins take part in virtually every biochemical process and play critical roles in nearly every cell structure. Cataloging the number and types of proteins present in an organism gives biochemists important insight into its structures and operations. Venter’s team hopes that identifying the minimum genome will provide them with an understanding of life at its most fundamental level.

In their attempts to reach that target, Venter’s group has focused attention on a bacterium called Mycoplasma genitalium. This microbe has one of the smallest, if not the smallest, genome known to scientists. M. genitalium parasitizes the human genital and respiratory tracts. Its genome possesses about 480 gene products. Because its genome is so extensively pared-down, it’s ideally suited as a model system to determine the absolutely indispensable requirements for life—the “non-negotiable” biochemical systems that must be present for an entity to be recognized as a form of life.

The researchers reasoned that it is quite likely that the bare essential genome is much smaller than 480 gene products. It turns out that a significant fraction of this parasite’s genome is dedicated to mediating interactions between the parasite and its host and can be considered as nonessential to a strictly minimal life-form.

Using an experimental approach, Venter’s team worked to ascertain the minimum number of genes needed for life. Their protocols involved both the random and systematic mutation of M. genitalium genes to determine those that are indispensable for life. (Biochemists refer to these procedures as knock-out experiments.) If a gene is nonessential, M. genitalium will still grow after the gene is mutated.

Once the essential or minimum gene has been determined via the knock-out experiments, the scientists hope to confirm their result by preparing a synthetic minimal genome and introducing it into a cell to see if the cell with the transplanted genome grows. They realized that in the process of identifying the minimum genome, they came just a few short steps away from making artificial life in the lab.

Steps to Creating Artificial Life

Venter and collaborators’ approach to creating an artificial life-form is called a top-down strategy. It involves starting with a naturally occurring microbe and stripping it down to its bare genetic and biochemical essence, and then modifying it by adding nonnative genes to the minimal genome to generate a nonnatural form of life.

The major stages in this effort involve:

systematically eliminating genes from the M. genitalium genome to identify all the essential genes;

synthesizing the building blocks of DNA from the minimal genome from nucleotides,;

introducing the minimal genome into the cytoplasm of a M. genitalium cell that has had its original genome deleted;

growing and then replicating the organism harboring the synthetic genome.

Once these steps are accomplished, a nonnatural organism, called Mycoplasma laboratorium, will have been created. At this point, the researchers will have the genetic foundation in place to build an organism with any biological properties they desire. For example, they currently plan on adding genes to the minimal genome that will produce proteins that can generate hydrogen.

A Brief Progress Report

To date Venter’s team has made remarkable progress toward their goal of producing Mycoplasma laboratorium. They have identified the essential gene set (which consists of about 380 genes). They have also synthesized from scratch the entire genome of a wild-type M. genitalium and cloned it (made copies) in yeast. Additionally, they have transferred the wild-type genome of M. genitalium into a closely related Mycoplasma species.

The next step is for the researchers to put all of these steps together to synthesize, clone, and introduce a synthetic minimal genome into M. genitalium.

Though this last milestone seems rather trivial, it is technically quite challenging. Next week I’ll illustrate these technical challenges by describing their most recent efforts to improve upon the efficiency of synthesizing and cloning a synthetic genome in yeast, a vital step in “bestowing animation on lifeless matter.”