Synthetic biology is defined as the de novo design and construction of biological devices and systems,1 and as such is heavily reliant on the use of synthetic DNA. The size and sequence of potential DNA constructs, however, have been hindered by the limitations of the two major techniques upon which synthetic biology is based: the amplification of cloned DNA copies using polymerase chain reaction (PCR) and E. coli, and the generation of recombinant DNA.
Methods that overcome these limitations are shaping the future of molecular biology and genetics research. This article explains how some of the most important advances in generating long stretches of DNA are changing the field of synthetic biology, and how the science of “big DNA” is becoming an increasingly accessible technology.
Constructing the largest synthetic DNA sequence
Traditional restriction/ligation-based cloning methods severely limit the size of the construct that can be assembled, and this challenge is driving the development of new techniques in big DNA.
In 2004, researchers at the J. Craig Venter Institute (Rockville, MD) set about to produce a 583-kb DNA construct in the form of the bacterial genome of Mycoplasma genitalium. At 18× larger than anything else that had ever been published, researchers had to invent entirely new methods to assemble the DNA. Published in 2008, this was the first time a whole genome had been constructed.2
Moving forward to 2010, the group assembled the larger, 1.1-Mb Mycoplasma mycoides genome. This was, and still is, the largest synthetic DNA sequence that had ever been assembled.3 The M. mycoides genome was activated to produce the first synthetic cell, and this groundbreaking event made news worldwide.
Limitations of older cloning methods
The construction of the largest synthetic DNA sequence would not have been possible using restriction/ligation based cloning methods, a major limitation being the need for unique restriction enzyme sequences in the working DNA. With short constructs that are less than 1 kb, the researcher can easily add a sequence that is compatible with the plasmid and insert design. As the sequences for cloning get longer, however, the list of unique restriction sites that do not already occur and that will not fragment the target sequence becomes much shorter.
Further, the addition of nonendogenous restriction sites to the construct produces a synthetic sequence that is different from the wild type. These sequences may result in unintended functional effects. A construct was needed that would not create these variant sequences and leave them in the final assembly.
Streamlining DNA synthesis
The work flow for constructing large DNA assemblies was a complicated task, requiring:
- Cloning of multiple fragments
- Weeding out errors by sequencing
- Assembling small, error-free DNA segments into the final, large construct
- Sequencing one more time.
This time-intensive method necessitates multiple rounds of cloning and sequencing, adding considerably to the cost. The discovery of novel solutions for building sequences that are much closer to 100% pure, while bypassing intervening cloning and sequencing steps, is vital for driving assembly costs down.
New DNA assembly method
Developed by SGI-DNA (a subsidiary of Synthetic Genomics Inc., La Jolla, CA), the Gibson Assembly™ Method4 does not rely on specific sequences. After assembling and amplifying 60-mer DNA oligos into 2–3 kb constructs, a proprietary enzyme mix was used for error correction. This was able to effectively degrade the majority of incorrect sequences, leaving a pool of very high-quality, assembled DNA.
The scientists then reamplified that population. In just a few hours, this strategy resulted in a population that is much faster to screen and that is enriched for the correct sequence. The method allows users to create, in less than one week, sequences between 10 and 30 kb that are the size of an entire biological pathway. Further, the user never needs to clone into E. coli. This technique is also faster than traditional cloning approaches, requiring only 1 hr for an entire assembly reaction.
The future of big DNA
With the ability to create longer DNA constructs, size is becoming less of a limitation for researchers, and the bottleneck has now shifted back to DNA designs. If users can easily build constructs of 30–100 kb that can constitute entire biological pathways all the way up to a whole bacterial genome, researchers need to start thinking about what they want to build.
Dr. Gibson and other scientists at the J. Craig Venter Institute are currently using these methods to understand what a minimal cell might look like. By building “minimal genomes” that include only the minimum elements required for a living cell, the team can identify what these genomes and cells need to look like. It will therefore provide an understanding of what every component within a cell is doing. The aim of this project is to accelerate gene design, providing a genetic framework to which users can add elements or modules. These modules can then be swapped in and out in order to test for functionality.
Dr. Gibson and his team are always looking for opportunities to refine and improve the DNA assembly methods that he and his colleagues have developed. They are also working on ways to improve large gene delivery to host cells, as well as methods that rely less on host organisms for copying their synthetic constructs.
A main focus is to make large DNA constructs more cost effective, and to develop manufacturing methods to commercialize such constructs. At this time, price remains a limiting factor—it still costs over $1 million to build a 1-Mb bacterial genome, which is cost prohibitive for many laboratories.
Dr. Gibson hopes eventually to be able to produce large assemblies for under one cent/base by taking advantage of the high quality and low cost of oligonucleotides from Integrated DNA Technologies, Inc. (IDT) (Coralville, IA), improved automation, and reduced DNA sequencing costs. Creating ways to decrease overall costs makes this technology far more accessible, opening the door to more research and even greater possibilities.
- Synthetic Biology: Industrial and Environmental Applications, 3rd ed.; Schmidt, M., Ed. Weinheim, Germany: Wiley–Blackwell, 2012, pp 1–67; ISBN 3-527-33183-2.
- Gibson, D.G.; Benders, G.A. et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 2008, 319(5867), 1215–20.
- Gibson, D.G.; Glass, J.I. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 2010, 329(5987), 52–6.
- Gibson, D.G.; Young, L. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Meth. 2009, 6(5), 343–45.
Dr. Dan Gibson, Ph.D., is an Associate Professor at the J. Craig Venter Institute, Rockville, MD, and Vice President of SGI-DNA, Inc., a subsidiary of Synthetic Genomics Inc., La Jolla, CA, U.S.A. Dr. Hans Packer, Ph.D., is Scientific Communications Writer, Integrated DNA Technologies, Inc. (IDT), 1710 Commercial Park, Coralville, IA 52241, U.S.A.; e-mail: firstname.lastname@example.org.