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Giant Golden Gate
To understand how genomes work, some scientists design and build new ones — a process that can take months or years. Now, an improved version of an existing technique has drastically increased the number of DNA pieces that can be stitched together at once, opening the door for small, designer genomes built in a single day.
In 2010, the J. Craig Venter Institute reported the first self-replicating, synthetic bacterial cell. In that case, the organism’s genome was created with chemistry; its DNA was joined together slowly, piece by piece. The project cost millions of dollars and took several years to complete, but it helped researchers better understand how DNA sequences, and the order of genes, affect the way an organism looks or behaves.
Ten years on, it’s easier than ever to build synthetic genomes. In a new study, researchers at New England Biolabs, a private company in Ipswich, Massachusetts, assembled 35 distinct fragments of DNA in one test tube. That’s a big improvement over existing methods for joining together DNA, which were typically limited to a handful of genetic fragments. The results were published in September in the journal PLOS ONE. But the researchers behind this work tell me that their method is already outdated.
The same team has now reported the assembly of an entire bacteriophage genome in a test tube from 52 distinct pieces of DNA. The results appeared in ACS Synthetic Biology this week, and were also revealed by Greg Lohman, senior scientist at New England Biolabs and lead author of the initial study, at the virtual SynBioBeta conference last year.
A bacteriophage is a type of virus that infects bacteria, and the genome assembled by Lohman’s team contains 40,000 nucleotides, or letters; by comparison, the human genome has over 3 billion letters. Still, the small genome is an impressive feat.
The method used by Lohman’s team to build the bacteriophage genome is called Golden Gate Assembly. The technique leverages molecular machines, called proteins, to chew up the ends of each DNA piece. The proteins leave behind “sticky” ends, which can then be joined to other pieces with complementary ends. To join 52 pieces together, the researchers meticulously tested, tweaked, and tuned those sticky ends until they identified an optimal combination. (In my prior research, I often used Golden Gate to stitch together about 12 pieces of DNA in one tube; Lohman’s achievement blows that out of the water.)
Building a synthetic bacteriophage genome is just a proof of concept for now, but the underlying method that made this achievement possible could be used to create any number of small, designer genomes. Several synthetic biologists agreed that Lohman’s research is significant. Just a few years ago, assembling DNA fragments was complicated, even for experts.
“When I was a first year PhD student, one of my rotation projects was to make a construct with three [DNA fragments],” said Naomi Nakayama, a researcher at Imperial College London who was not involved in the study. “I worked on it for three months.”
Oh, how times have changed.
Adapted from my prior article for Scienceline.
Read more at ACS Synthetic Biology.
(↑ = recommended article, * = open access, † = review article )
↑*Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous systems. Chen X…Gradinaru V. Neuron. Link
An autonomously oscillating supramolecular self-replicator. Howlett MG…Fletcher SP. Nature Chemistry. Link
*Unifying the known and unknown microbial coding sequence space. Vanni C…Fernàndez-Guerra A. eLife. Link
*Growth-dependent heterogeneity in the DNA damage response in Escherichia coli. Jaramillo-Riveri S…El Karoui M. Molecular Systems Biology. Link
*Traditional Protocols and Optimization Methods Lead to Absent Expression in a Mycoplasma Cell-Free Gene Expression Platform. Sakai A…Glass JI. Synthetic Biology. Link
↑Biosynthesis of cyanobacterin, a paradigm for furanolide core structure assembly. D’Agostino PM…Gulder TAM. Nature Chemical Biology. Link
*Enzymatically amplified linear dbDNATM as a rapid and scalable solution to industrial lentiviral vector manufacturing. Barreira M…Smith-Moore S. Gene Therapy. Link
*Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols. Bean BDM…Martin VJJ. Nature Communications. Link
*Integration of metabolism and regulation reveals rapid adaptability to growth on non-native substrates. Trivedi VD…Nair NU. bioRxiv (preprint). Link
*Improving recombinant protein production by yeast through genome-scale modeling using proteome constraints. Li F…Nielsen J. Nature Communications. Link
↑*Complex dynamics in a synchronized cell-free genetic clock. Aufinger L, Brenner J & Simmel FC. Nature Communications. Link
*Cooperative assembly confers regulatory specificity and long-term genetic circuit stability. Bragdon MDJ…Khalil AS. bioRxiv (preprint). Link
Computational Tools & Models
*Prediction of protein–ligand binding affinity from sequencing data with interpretable machine learning. Rube HT…Bussemaker HJ. Nature Biotechnology. Link
*A framework to efficiently describe and share reproducible DNA materials and construction protocols. Mori H & Yachie N. Nature Communications. Link
*AutoESD: a web tool for automatic editing sequence design for genetic manipulation of microorganisms. Yang Y…Ma H. Nucleic Acids Research. Link
*FABIAN-variant: predicting the effects of DNA variants on transcription factor binding. Steinhaus R, Robinson PN & Seelow D. Nucleic Acids Research. Link
*PepNN: a deep attention model for the identification of peptide binding sites. Abdin O…Kim PM. Communications Biology. Link
CRISPR & Genetic Control
CRISPR/dCas9-RpoD-Mediated Simultaneous Transcriptional Activation and Repression in Shewanella oneidensis MR-1. Chen Y…Cao Y. ACS Synthetic Biology. Link
*A Well-Characterized Polycistronic-Like Gene Expression System in Yeast. Mukherjee M & Wang ZQ. bioRxiv (preprint). Link
*CRISPR base editing of cis-regulatory elements enables target gene perturbations. Lim CKW…Gaj T. bioRxiv (preprint). Link
*Targeted directional kilobase sequence insertion by combining prime editing with recombinases or integrases. Jillette N, Zhu JJ & Cheng AW. bioRxiv (preprint). Link
*Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells. Haideri T…Lian XL. bioRxiv (preprint). Link
Medicine & Diagnostics
A vaccine targeting resistant tumours by dual T cell plus NK cell attack. Badrinath S…Wucherpfennig KW. Nature. Link
↑*Onasemnogene abeparvovec gene replacement therapy for the treatment of spinal muscular atrophy: a real-world observational study. Bitetti I…Varone A. Gene Therapy. Link
*Synthetic gene circuits for preventing disruption of the circadian clock due to interleukin-1–induced inflammation. Pferdehirt L…Guilak F. Science Advances. Link
*Automated amplification-free digital RNA detection platform for rapid and sensitive SARS-CoV-2 diagnosis. Shinoda H…Watanabe R. Communications Biology. Link
Temporal Expression of Transcription Factor ID2 Improves Natural Killer Cell Differentiation from Human Pluripotent Stem Cells. Jung J…Bao X. ACS Synthetic Biology. Link
Effects of intracardiac delivery of aldehyde dehydrogenase 2 gene in myocardial salvage. Pan G…Palaniyandi SS. Gene Therapy. Link
*Using Extracellular Vesicles Released by GDNF-transfected Macrophages for Therapy of Parkinson’s Disease. Zhao Y…Batrakova EV. bioRxiv (preprint). Link
†Gene Therapy in Orthopaedics: Progress and Challenges in Pre-clinical Development and Translation. Ghivizzani SC…Evans C. Frontiers in Bioengineering and Biotechnology. Link
*Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells. Omer-Javed A…Naldini L. Cell. Link
*Retrieving a Disrupted Gene Encoding Phospholipase A for Fiber Enhancement in Allotetraploid Cultivated Cotton. Fang L…Zhang T. Plant Biotechnology Journal. Link
†The integrated genomics of crop domestication and breeding. Huang X…Li J. Cell. Link
Protein & Molecular Engineering
Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex. Kato…Nishimasu H. Cell. Link
*Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice. Hunt AC…Baker D. Science Translational Medicine. Link
*Design of Peptide-Based Protein Degraders via Contrastive Deep Learning. Palepu K…Koseki SRT. bioRxiv (preprint). Link
A fluorescent nanosensor paint detects dopamine release at axonal varicosities with high spatiotemporal resolution. Elizarova S…Daniel JA. PNAS. Link
*Single-domain near-infrared protein provides a scaffold for antigen-dependent fluorescent nanobodies. Oliinyk OS…Verkhusha VV. Nature Methods. Link
Tools & Technology
*Rewritable two-dimensional DNA-based data storage with machine learning reconstruction. Pan C…Milenkovic O. Nature Communications. Link
*A super robust and efficient DNA storage architecture based on modulation encoding and decoding. Zan X…Liu W. bioRxiv (preprint). Link
Functional Nanopore Screen: A Versatile High-Throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli. Weber W…Stein V. ACS Synthetic Biology. Link
*An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae. Peng B…Vickers CE. Nature Communications. Link
↑*3D printed protein-based robotic structures actuated by molecular motor assemblies. Jia H…Schwille P. Nature Materials. Link