This Small Business Innovation Research (SBIR) Phase I project attempts to radically reduce the cost of error-free oligonucleotides for use with gene synthesis. DNA Synthesis of large molecules is done by the assembly of many short oligonucleotide fragments of DNA 60-100bp in length. Currently, each DNA fragment is synthesized in relatively small numbers at an excessive macroscopic scale that incurs a large manufacturing overhead in its production costs. This sets a high cost floor for the entire DNA synthesis process. One route to making cheaper oligonucleotides is to synthesize them in large sets on microarrays. However, array-synthesized DNA is both extremely error-prone and produced as dilute, complex mixtures. This proposed project will use massively parallel sequencers to sequence clonally amplified copies of DNA species sampled from the microarray in order to sort every species apart from one another as well as to identify correctly synthesized oligonucleotides from incorrect ones. Further, it is proposed to use focused laser pulses in a custom laser ejection device to eject and recover desired subsets of perfect oligos from micron-scale sequenced colonies into multiwell plates for assembly into genes. The goal is to be able to recover tens of thousands of sequence-verified oligonucleotides in several hours from sequencer flowcells.
The broader impact/commercial potential of this project is to achieve truly disruptive cost decreases in the DNA synthesis of arbitrary genetic information. With the abundance of sequencing data, it is possible to imagine entering a new era of "constructive" biology, where in addition to classical reductive experiments on components, it will be possible to test our understanding of genetic- and protein-based circuits by synthesizing new designs and measuring their discrepancy from predicted behaviors. The low-cost industrial production of arbitrary synthetic DNA has the potential to change the practice of biology such that it becomes cost effective to engineer whole genetic pathways and even genomes, accelerating the development of bioengineering, synthetic biomaterials production, as well as medical and research applications.
Our understanding of biology has advanced rapidly in recent years as the complexities of the genetic code have begun to be unraveled with rapid progress in high-throughput DNA sequencing. Only recently has there been the capability to sequence complete human genomes and understand the impacts on health, disease, and function. A parallel technology that is intimately related to DNA sequencing is synthetic biology, a field that aims to write programmatic functions into cells through genetic modifications. Unfortunately, despite great potential in the uses of synthetic biology, the field is bottlenecked by the high cost of synthetic DNA. This high cost puts pressure on the researchers to either handcraft genes themselves, a time consuming and error prone task, or severely limit the number of gene orders they make. The goal of our SBIR Phase I Project was to develop a massively-parallel synthesis pipeline for synthetic DNA. Currently, gene synthesis is performed by assembling smaller DNA fragments, each of which is synthesized one at a time at an excessive macroscopic scale. This is not only a slow and laborious process, but an expensive one and therefore retail gene DNA is often prohibitively expensive for rapid research and design iterations. Cambrian Genomics aims to solve this problem by creating DNA with a massively parallel pipeline that drastically reduces synthetic DNA cycle time at a price point an order of magnitude cheaper than is currently available. We have developed a method for taking highly complex and imperfect mixtures of synthetic DNA fragments produced at massive scale on microarrays and purifying out only the perfectly made pieces into precise combinations suited for assembly into synthetic genes. We have achieved this by copying the DNA onto a micron-sized beads that we then proof-read using next-generation sequencing technology, then selecting only those perfect beads by laser-ejection into multi-well plates for enzymatic assembly. During the course of this SBIR project, we have developed custom modular flowcells and software for binding these DNA-bearing beads, sequencing them, then planning a route for a high-powered laser to eject specific beads for capture and amplification by PCR. Using these, we have demonstrated recovery and assembly of simple DNA fragments using our laser-selection process.
