<p>Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize and screen in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis and error correction technology has far lagged behind that of gene and genome sequencing. What even lagged behind is the capability of screening a large population of information on a single cell, protein or gene level. Compartmentalization of single cells in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with quantitative readouts. However these single-emulsion droplets are incompatible with aqueous phase analysis and are not controllable through molecule transports. </p><p>This thesis presents the development of a multi-tool ensemble platform targeted at high-throughput gene synthesis, error correction and screening. An inkjet oligonucleotide synthesizer is constructed to synthesize oligonucleotides as sub-arrays onto patterned and functionalized thermoplastic microchips. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. Bacterial cells baring individual synthetic gene variants are encapsulated as single cells into double-emulsion droplets where cell populations are enriched by up to 1000 times within several hours of proliferation. Permeation of Isopropyl-D-1-thiogalactopyranoside (IPTG) molecules from the external solution allows induction of target gene expression. The induced expression of the synthetic fluorescent proteins from at least ~100 bacteria per droplet generates clearly distinguishable fluorescent signals that enable droplets sorting through fluorescence-activated cell sorting (FACS) technique. The integration of oligo synthesis and gene assembly on the same microchip facilitates automation and miniaturization, which leads to cost reduction and increases in throughput. The capacity of double emulsion system (millions discrete compartments in 1ml solution) combined with high-throughput sorting by FACS provide the basis for screening complex gene libraries for different functionality and activity, significantly reducing the cost and turn-around time.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/9801 |
| Date | January 2015 |
| Creators | Ma, Siying |
| Contributors | Yuan, Fan, Tian, Jingdong |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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