Abstract

The rapid manufacturing of highly accurate synthetic DNA is crucial for its use as a molecular tool, the understanding and engineering of regulatory elements, protein engineering, genetic refactoring, engineered genetic networks and metabolic pathways, and whole-genome syntheses [1,2]. Recent efforts in the development of enzyme mediated oligonucleotide synthesis have shown much potential to benefit conventional DNA synthesis [3–5]. With its success in applications as chemical microreactors [6–10], biological assays [8–14], and clinical diagnostic tools [10,15–19], digital microfluidic (DMF) devices are an attractive platform to apply the promising benefits of enzymatic oligonucleotide synthesis to the manufacturing of synthetic DNA. This thesis work aims to demonstrate automated DNA assembly using oligonucleotides on a DMF device through the demonstration and validation of an automated DNA assembly protocol.

The prototyping process performed through this work revealed various important design considerations for the reliability of fluid handling performance and the mitigation of failure modes. To prevent dielectric breakdown or electrolysis, a relatively thick SU-8 3005 dielectric is used to remove the sensitivity of the device to variances in dielectric thickness and quality. To enable droplet creation, the gap distance between the DMF chip and top-plate is created and minimized using a thick SU-8 2100 layer. Reliable droplet creation is achieved through the use of electrode geometry that targets predictable fluid delivery and cutting. Reliable droplet transport is achieved through the use of a electrode interdigitation geometry that targets lower total electrode surface area and higher interdigitation contact area. The testing of DNA laden fluids revealed that biofouling can be a large concern for the demonstration of DNA assembly on a DMF device if droplets are moved through an air medium. To mitigate its effects, the final DMF device design featured the use of a permanently bonded top-plate with bored inlet/outlet ports as well as a silicone oil medium.

The final DMF device design was used to demonstrate automated DNA assembly. This demonstration involved the creation, transport, and mixing of DNA brick samples. These samples are subsequently incubated on a chemical bench or on the DMF chip to create recombinant DNA containing genetic information. DNA gel imaging of DNA assembly products from on-chip protocols compared to protocols performed on a chemical benchtop revealed comparable results. Through the course of this work, the applicability of automated DNA assembly on a DMF device was validated to provide preliminary results in the ultimate goal of DNA synthesis using enzymatic oligonucleotide synthesis.

Library of Congress Subject Headings

DNA--Synthesis; Synthetic biology; Microfluidic devices; Oligonucleotides

Publication Date

5-12-2021

Document Type

Thesis

Student Type

Graduate

Degree Name

Microelectronic Engineering (MS)

Department, Program, or Center

Microelectronic Engineering (KGCOE)

Advisor

Michael Schertzer

Advisor/Committee Member

Karl Hirschman

Advisor/Committee Member

Ivan Puchades

Campus

RIT – Main Campus

Plan Codes

MCEE-MS

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