Making Artificial Bacteria
A team of 17 researchers at the J. Craig Venter Institute (JCVI) has created the largest man-made DNA structure by synthesizing and assembling the 582,970 base pair genome of a bacterium, Mycoplasma genitalium JCVI-1.0.
This work, published online in the journal Science by Dan Gibson, Ph.D., et al, is the second of three key steps toward the team’s goal of creating a fully synthetic organism. In the next step, which is ongoing at the JCVI, the team will attempt to create a living bacterial cell based entirely on the synthetically made genome.
The team achieved this technical feat by chemically making DNA fragments in the lab and developing new methods for the assembly and reproduction of the DNA segments. After several years of work perfecting chemical assembly, the team found they could use homologous recombination (a process that cells use to repair damage to their chromosomes) in the yeast Saccharomyces cerevisiae to rapidly build the entire bacterial chromosome from large subassemblies.
“This extraordinary accomplishment is a technological marvel,” said J. Craig Venter, Ph.D., President and Founder of JCVI. “Ham Smith, Clyde Hutchison, Dan Gibson, Gwyn Benders, and the others on this team dedicated the last several years to designing and perfecting new methods and techniques that we believe will become widely used to advance the field of synthetic genomics.”
The building blocks of DNA—adenine (A), guanine (G), cytosine (C) and thiamine (T) are not easy chemicals to artificially synthesize into chromosomes. As the strands of DNA get longer they get increasingly brittle, making them more difficult to work with. Prior to this study, the largest synthesized DNA contained only 32,000 base pairs. Thus, building a synthetic version of the genome of the bacteria M. genitalium genome that has more than 580,000 base pairs presented a formidable challenge. However, the JCVI team has expertise in many technical areas and a keen biological understanding of several species of mycoplasmas.
“When we started this work several years ago, we knew it was going to be difficult because we were treading into unknown territory,” said Hamilton Smith, M.D., senior author on the publication. “Through dedicated teamwork we have shown that building large genomes is now feasible and scalable so that important applications such as biofuels can be developed.”
Methods for Creating the Synthetic M. genitalium
The process to synthesize and assemble the synthetic version of the M. genitalium chromosome began first by resequencing the native M. genitalium genome to ensure that the team was starting with an error free sequence. After obtaining this correct version of the native genome, the team specially designed fragments of chemically synthesized DNA to build 101 “cassettes” of 5,000 to 7,000 base pairs of genetic code. As a measure to differentiate the synthetic genome versus the native genome, the team created “watermarks” in the synthetic genome. These are short inserted or substituted sequences that encode information not typically found in nature. Other changes the team made to the synthetic genome included disrupting a gene to block infectivity. To obtain the cassettes the JCVI team worked primarily with the DNA synthesis company Blue Heron Technology, as well as DNA 2.0 and GENEART.
From here, the team devised a five stage assembly process where the cassettes were joined together in subassemblies to make larger and larger pieces that would eventually be combined to build the whole synthetic M. genitalium genome. In the first step, sets of four cassettes were joined to create 25 subassemblies, each about 24,000 base pairs (24kb). These 24kb fragments were cloned into the bacterium Escherichia coli to produce sufficient DNA for the next steps, and for DNA sequence validation.
The next step involved combining three 24kb fragments together to create 8 assembled blocks, each about 72,000 base pairs. These 1/8th fragments of the whole genome were again cloned into E. coli for DNA production and DNA sequencing. Step three involved combining two 1/8th fragments together to produce large fragments approximately 144,000 base pairs or 1/4th of the whole genome.
At this stage the team could not obtain half genome clones in E. coli, so the team experimented with yeast and found that it tolerated the large foreign DNA molecules well, and that they were able to assemble the fragments together by homologous recombination. This process was used to assemble the last cassettes, from 1/4 genome fragments to the final genome of more than 580,000 base pairs. The final chromosome was again sequenced in order to validate the complete accurate chemical structure.
The synthetic M. genitalium has a molecular weight of 360,110 kilodaltons (kDa). Printed in 10-point font, the letters of the M. genitalium JCVI-1.0 genome span 147 pages.
“This is an exciting advance for our team and the field. However, we continue to work toward the ultimate goal of inserting the synthetic chromosome into a cell and booting it up to create the first synthetic organism,” said Dan Gibson, lead author.
The research to create the synthetic M. genitalium JCVI-1.0 was funded by Synthetic Genomics, Inc.
Background/Key Milestones in JCVI’s Synthetic Genomics Research
The work described by Gibson et al. has its genesis in research by Dr. Venter and colleagues in the mid-1990s after sequencing M. genitalium and beginning work on the minimal genome project. This area of research, trying to understand the minimal genetic components necessary to sustain life, began with M. genitalium because it is a bacterium with the smallest genome that we know of that can be grown in pure culture. That work was published in the journal Science in 1995.
In 2003 Drs. Venter, Smith and Hutchison made the first significant strides in the development of a synthetic genome by their work in assembling the 5,386 base pair bacteriophage ?X174 (phi X). They did so using short, single strands of synthetically produced, commercially available DNA (known as oligonucleotides) and using an adaptation of polymerase chain reaction (PCR), known as polymerase cycle assembly (PCA), to build the phi X genome. The team produced the synthetic phi X in just 14 days.
In June 2007 another advance was achieved when JCVI researchers led by Carole Lartigue, Ph.D., announced the results of work on genome transplantation methods allowing them to transform one type of bacteria into another type dictated by the transplanted chromosome. The work was published in the journal Science, and outlined the methods and techniques used to change one bacterial species, Mycoplasma capricolum, into another, Mycoplasma mycoides Large Colony (LC), by replacing one organism’s genome with the other one’s genome.
Genome transplantation was the first essential enabling step in the field of synthetic genomics as it is a key mechanism by which chemically synthesized chromosomes can be activated into viable living cells. This announcement of the successful synthesis of the M. genitalium genome is the second step leading to the next experiments to transplant a fully synthetic bacterial chromosome into a living organism and “boot up” the cell.
Ultimately, this method of ‘building’ a cell from the ground up can teach us a great deal about the fundamentals of life. Understanding the minimal requirements needed to create functional DNA and, ultimately, a living cell can help astrobiologists determine the conditions necessary for life to originate on Earth and other locations in the Universe.