Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening
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1.
California Institute of Technology
Abstract
A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.
Copyright and License
© 2024 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0.
Acknowledgement
The authors would like to thank Rohit Nayak for providing calibration data for the US transducer used for imaging. Transmission electron microscopy was done in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. This research was supported by the National Institutes of Health (R01-EB018975 to M.G.S.), the Chan-Zuckerberg Initiative and Pew Charitable Trust. R.C.H. was supported by the Caltech Center for Environmental Microbial Interactions. Related research in the Shapiro Laboratory is supported by the David and Lucile Packard Foundation. M.G.S. is an investigator of the Howard Hughes Medical Institute.
Contributions
R.C.H. and Z.J. contributed equally to this work. R.C.H., Z.J., and M.G.S. conceived and designed the study. Z.J. and D.R.M. designed and built the Acoustic Plate Reader hardware. Z.J., D.P.S., and D.R.M. wrote the MATLAB scripts for data acquisition with the Acoustic Plate Reader. R.C.H., Z.J., and D.P.S. wrote the MATLAB scripts for data analysis from the Acoustic Plate Reader. M.S. designed the MATLAB graphical user interfaces for Acoustic Plate Reader data acquisition and analysis. R.C.H., M.S., K.W., H.K.S., R.D., and R.Z. performed directed evolution experiments. P.D. performed TEM imaging. R.C.H. analyzed all data. R.C.H. wrote the paper, with input from all authors. M.G.S. supervised the research.
Data Availability
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Supplementary Video 1: Example acoustic plate reader scan. (Right) The Acoustic Plate Reader is scanning six 96-well phantoms; (left) the computer screen displays the real-time images of linear (left) and nonlinear (middle) contrast, as well as the Verasonics control interface (right) (MP4)
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Table S1: Oligos used for mutagenesis. Sequences of the oligos that composed the four oligo pools used to create the GvpA/GvpB libraries. “Library Round” indicates the round of screening (first or second) in which the oligo was used, and “Sub-Library” indicates the pool in which it was synthesized; Table S2: Custom-made MoClo parts. Inventory of the MoClo parts added to the base EcoFlex system and used for cloning the constructs in this study; Table S3: Ultrasound pulse sequences. List of the parameters entered into the APR GUI to perform each scan in this study; Table S4: PCR primers. Sequences of the primers used to either amplify the oligo pools that were used to create the libraries, or to reclone the best gvpA/gvpB mutants into Level 0 MoClo part vectors for assembly into expression constructs and subsequent validation (XLSX)
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Detailed diagram of the Acoustic Plate Reader workflow; details of gvpA/gvpB mutant library construction; characterization of the top mutants from Rounds 1 and 2 of evolution; acoustic collapse pressure curves for the best mutants identified in this study; TEM images of E. coli cells expressing WT or mutant A. flos-aquae GVs; TEM images of E. coli cells expressing WT or mutant B. megaterium GVs; Golden Gate reaction parameters (PDF)
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Code S1: MATLAB script and associated files for generating the scanning site saturation libraries (ZIP)
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Code S2: MATLAB script and associated files for generating the recombination library (ZIP)
Conflict of Interest
The authors declare no competing financial interest.
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Additional details
- ISSN
- 2161-5063
- National Institutes of Health
- R01-EB018975
- Chan-Zuckerberg Initiative
- Pew Charitable Trusts
- California Institute of Technology
- Caltech Center for Environmental and Microbial Interactions
- David and Lucile Packard Foundation
- Howard Hughes Medical Institute
- Caltech groups
- Caltech Center for Environmental Microbial Interactions (CEMI)