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Engineering of Second-Generation Acoustic Reporter Genes


Hurt, Robert Cooper (2023) Engineering of Second-Generation Acoustic Reporter Genes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/qs6v-5d67.


A major outstanding challenge in the fields of biological research, synthetic biology, and cell-based medicine is visualizing the functions of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 µm. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles (GVs) to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo.

The goal of my thesis work has been to engineer second-generation ARGs with improved acoustic and expression phenotypes compared to the existing first-generation constructs. I took two complementary engineering approaches to developing these constructs: homolog screening and directed evolution, sometimes referred to as the “nature and nurture” of protein engineering. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue in vivo, stable long-term expression, and compatibility with in vitro multiplexed imaging. In collaboration with others in the lab, we demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies.

This thesis is organized as follows: in the first two chapters, I introduce the key background needed to understand both the importance and properties of ARGS, and how they have been and could be engineered. In the next two chapters, I detail specific efforts to engineer them—one involving the construction of a high-throughput, semi-automated setup for acoustic phenotyping of cells and its application to ARG directed evolution, and another involving the screening of several GV cluster homologs to identify ones suitable for use as improved ARGs. Finally, I conclude with insights gleaned from these two ARG engineering projects and suggestions for future ones.

The approaches, results, and ideas presented in this thesis represent the current state-of-the-art in ARG engineering and application. While recent technology development in this field has unlocked exciting new use cases for ARGs in noninvasive biological imaging, most of their potential for basic science and disease diagnosis and treatment has yet to be realized.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Gas vesicles, Acoustic Reporter Genes, ultrasound, protein engineering, synthetic biology
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Neurobiology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Shapiro, Mikhail G.
Thesis Committee:
  • Lester, Henry A. (chair)
  • Murray, Richard M.
  • Rees, Douglas C.
  • Shapiro, Mikhail G.
Defense Date:23 March 2023
Non-Caltech Author Email:robertchurt (AT)
Record Number:CaltechTHESIS:05192023-001330664
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Chapter 4.
Hurt, Robert Cooper0000-0002-4347-6901
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15194
Deposited By: Robert Hurt
Deposited On:26 May 2023 22:31
Last Modified:08 Nov 2023 00:33

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