Electromagnetic Tweezer Development: Improving Single-Molecule Techniques for the Study of DNA Mechanics and Gene Transcription Open Access

Piccolo, Joseph (Fall 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/m039k598h?locale=en


DNA transcription is the foundational biological process to all cellular life. Transcription and eventual gene expression are regulated via proteins known as transcription factors. DNA torsion and tension play an important role in the binding and unbinding of transcription factors. The interplay between these physical parameters and transcription factors is key to the understanding of transcriptional regulation; yet, it is extremely hard to study in vivo. Bulk in vitro measurements are the averages of an Avogadro’s number scale of molecules, a scope that obscures individual molecular details. Thus, single-molecule techniques have emerged as powerful approaches to study molecular mechanisms. A magnetic tweezer is a single molecule technique which allows application of tension and torque to individual DNA molecules in solution. The most common setup, a pair of permanent rare-earth magnets, is positioned near the sample, above an optical microscope stage using motorized mechanical translators. Unfortunately, the electric motor introduces unwanted vibrations into the system, reducing the accuracy of measurements. This thesis describes the development of an electromagnetic tweezer that eliminates physical motion within the optical system and includes custom electromagnetic solenoids, control electronics, as well as ad hoc software. The ability of these electromagnetic tweezers to stretch and twist DNA was tested in preliminary measurements and possible applications of this instrument in the study of transcription and its regulation are discussed.

Table of Contents

Table of Contents

I.   Introduction

A.      DNA and The Central Dogma

B.      DNA Supercoiling

C.      Single Molecule Experimentation/DNA Tethers

D.      Magnetic Tweezers

E.      Solenoid Magnetic Fields

II.      Electromagnet Construction

A.      System Overview

B.      Frame and Solenoid Construction

C.      Electrical Hardware

D.      Computer Software and Interface Structure

III.     Experimental Planning

A.      DNA Sample Preparation

B.      Flow-chamber Preparation

C.      Particle Tracking and Microscopy

D.      Force Calculations

E.      Testing the Electromagnet

IV.     Results and Discussion

A.      Force Extension Test

B.      Extension vs. Turns Test

C.      Stepwise Tests

D.      Future Aims

V.      Works Cited

VI.     Appendix

A.      pDD_IN2BbvCI Plasmid

B.      Force Extension Software

C.      Extension vs. Turns Software

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