Biophysics

* These authors contributed equally to the work.O’Connor, C.*, Sheneman, A.*, Ferguson, M., & Herne, C. (2024). "Measuring attachment rate of predatory bacteria to prey using optical tweezers." Optical Trapping and Optical Micromanipulation. Conference Paper. 10.1117/12.2675569O’Connor, C.*, Sheneman, A. C.*, Ferguson, M., & Herne, C. (2023). "Measuring Time from Attachment of B. bacteriovorus to Prey." Student Research Symposium at New Paltz University. Poster.

CONFERENCE PAPER

ResearchGate Publication Link or DOI:10.1117/12.2675569

Overview

New Paltz University Research ('22-'23)Mentor: Prof. Catherine Herne, Department of Physics & Astronomy at New Paltz UniversityProf. Megan Ferguson, Department of Chemistry at New Paltz UniversitySummary: Assessed bacterial attachment rates using optical tweezers to investigate antibiotic resistance. Results: Demonstrated that predatory bacterial attachment via Type IV pili on prey E. coli occurs significantly faster than previously understood.

Methods

Antibiotic resistance is a growing issue. This project addresses how the predatory Bdellovibrio bacteriovorus may be a useful antibiotic. These predatory bacteria use type IV pili to form an attachment to their prey, Escherichia coli for the purposes of this project. Utilizing optical tweezers, we can position the bacteria to facilitate for these attachments and determine the rates at given time intervals. This allows us to further examine the time it takes for the initial attachment to form. We conclude that the attachment is significantly faster than previously understood, indicating that type IV pili may perform almost instantaneous attachments once in contact with their prey bacteria. We additionally predict that the attachment rates would tend toward zero as the time increments decrease, though further studies at short time intervals are needed.

Image from the optical tweezers demonstrating the appearance of E. coli and B. bacteriovorus at 5 micrometers

A cross-section of the slide layout. The top is the slide, then there is a section sandwiched between tape that reads from top to bottom as Cell-Tak, E. coli, B. bactriovorus. Next is the coverslip that rests on the tape and immersion oil on the opposite side of the coverslip. The slide is facing down as it would be in the optical tweezers.

Bacteria in the Optical Tweezers

Images at two varying depths retrieved from our optical tweezers. Each demonstrate an Escherichia coli as oblong large rods with an attached Bdellovibrio bacteriovorus indicated by the arrows. The difference is color is based on the focal point of the light as it moves through the slide at varying depth.

Slide Set-Up

Cell-tak was used as an adhesive on a plasma cleaned glass slide to attach Escherichia coli. The E. coli were then stationary targets. The Bdellovibrio Bacteriovorus were then captured from their solution in the beam waist of the laser focus. The B. Bacteriovorus were free-floating in solution beneath the cover slip.

In this project, I was responsible for every component of the research: plasma cleaning microscope slides, propagating the bacteria, making the setup as pictured above, utilizing the optical tweezers to measure attachments, and analyzing the data.

Results

The attachment percentages were determined from over 200 trials at 15 seconds, 30 seconds, 1 minute, and 2 minutes. The latter three fell within the confidence interval of an approximately 50% attachment rate whereas the 15 second trials were distinctly lower. This is orders of magnitude quicker than previously understood.The 50% rate is possibly based on the orientation of the bacteria's pili when trapped within the laser (facing the prey or facing away).