Update on designing and building minimal cells
Introduction
Design-based engineering of biological systems (also known as synthetic biology) tests understanding of the living world and harnesses its diverse repertoire to solve society's problems [1, 2]. Ideally, an engineered system should be functionally robust and predictable. Yet these features are difficult to achieve when engineering biology [3] because of the poorly understood complexity of even the simplest single-celled organisms. An enticing way to simplify cellular complexity, test understanding, and potentially facilitate engineering is to synthesize minimal cells [4, 5, 6, 7]. Forster and Church reviewed plans of others to minimize small bacterial cells (in vivo ‘top-down’ approach) [5] and proposed detailed plans for synthesizing a minimal cell from biomolecular parts (in vitro ‘bottom-up’ approach) [4]. Here, we highlight progress, challenges, and prospects since these two reviews.
Section snippets
New tools
Minimal cells require minimal genomes, and minimal genomes require design, construction, and manipulation tools at an unprecedented scale. Great progress has been made in genome construction by the J. Craig Venter Institute (JCVI; Rockville, MD, USA). JCVI constructed the 582 kilobase pair (kbp) genome of Mycoplasma genitalium, the smallest known genome of a bacterium capable of independent growth [8]. This was done by commercial gene synthesis from oligodeoxyribonucleotides (oligos) and then
Top-down approach: in vivo reduction
Even the most highly reduced genome of M. genitalium contains 100 individually dispensable genes out of 528 annotated genes [20], so streamlining down to only essential genes is one route to minimal cells. So far, significant minimization has been carried out only in organisms with larger genomes such as E. coli (4640 kbp; 4434 genes) and Bacillus subtilis (4216 kbp; 4245 genes) aided by known sequences of closely related genomes. Genome reduction by up to 30% has proven surprisingly successful
Bottom-up approach: in vitro construction
The alternative direction to a minimal cell is bottom-up: synthesizing self-replication by pooling together essential purified biological macromolecules, their genes, and their small molecule substrates [4]. By this approach, cellular overhead including genes of unknown function can be removed, the system can be readily manipulated and tuned, and all of the components can be defined. One possibility is a DNA/RNA/protein system derived from the core replication machinery of today's simplest
Prospects for biotechnology
Minimal cell syntheses are still in their formative stages where the main rewards are new molecular tools and a better understanding of the core genetic and biochemical systems necessary for basic life. But applications in biotechnology are close at hand. Based on the improved stability, growth, and protein production of E. coli and other biotechnology workhorses upon reducing their genomes [21••, 22, 23], further minimized strains should replace most current commercial bacterial strains.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We are grateful to George Church for advice and comments on the manuscript and John Glass, John McCutcheon, and Michael Sismour for comments on the manuscript. This work was supported by the National Institutes of Health and National Academies Keck Futures Initiative (to MCJ and ACF), the National Science Foundation (to MCJ), and the American Cancer Society (to ACF).
References (70)
- et al.
Chemical approaches to synthetic biology
Curr Opin Biotechnol
(2009) - et al.
Transcriptome complexity in a genome-reduced bacterium
Science
(2009) Reconstitution of ribosomes
- et al.
The weird and wonderful world of bacterial ribosome regulation
Crit Rev Biochem Mol Biol
(2007) - et al.
A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit
Nucleic Acids Res
(2009) - et al.
Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis
Biotechnol Bioeng
(2004) - et al.
Enhanced D-amino acid incorporation into protein by modified ribosomes
J Am Chem Soc
(2003) - et al.
Cell-free translation reconstituted with purified components
Nat Biotechnol
(2001) - et al.
The second wave of synthetic biology: from modules to systems
Nat Rev Mol Cell Biol
(2009) - et al.
Microbial production of fatty-acid-derived fuels and chemicals from plant biomass
Nature
(2010)