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Genomically Mined Acoustic Reporter Genes Enable On-Demand In Vivo Monitoring of Tumor-Homing Bacteria

Hurt, Robert C. and Buss, Marjorie T. and Wong, Katie and Sawyer, Daniel P. and Swift, Margaret B. and Dutka, Przemysław and Mittelstein, David R. and Jin, Zhiyang and Abedi, Mohamad H. and Deshpande, Ramya and Shapiro, Mikhail G. (2021) Genomically Mined Acoustic Reporter Genes Enable On-Demand In Vivo Monitoring of Tumor-Homing Bacteria. . (Unpublished)

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A major outstanding challenge in the fields of biological research, synthetic biology and cell-based medicine is the difficulty of visualizing the function 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 a spatial resolution below 100 um. Recently, the first genetically encoded reporter molecules were developed based on bacterial gas vesicles 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 in the in vivo context. Here, we describe second-generation ARGs with greatly improved acoustic properties and expression characteristics. We identified these ARGs through a systematic phylogenetic screen of candidate gas vesicle gene clusters from diverse bacteria and archaea. The resulting constructs offer major qualitative and quantitative improvements, including the ability to produce nonlinear ultrasound contrast to distinguish their signals from those of background tissues, and a reduced burden of expression in probiotic hosts. We demonstrate the utility of these next-generation ARGs by imaging the in situ gene expression of tumor-homing probiotic bacteria, revealing the unique spatial distribution of tumor colonization by these cells noninvasively in living subjects.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Hurt, Robert C.0000-0002-4347-6901
Buss, Marjorie T.0000-0002-4266-9197
Sawyer, Daniel P.0000-0003-2926-191X
Swift, Margaret B.0000-0001-9610-0687
Dutka, Przemysław0000-0003-3819-1618
Mittelstein, David R.0000-0001-8747-0483
Jin, Zhiyang0000-0002-4411-6991
Abedi, Mohamad H.0000-0001-9717-6288
Shapiro, Mikhail G.0000-0002-0291-4215
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This version posted April 27, 2021. The authors would like to thank Dianne Newman for a sample of Streptomyces coelicolor A3(2), and Yunfeng Li and Avinoam Bar-Zion for help with tissue histology. Electron microscopy was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. R.C.H. was supported by the Caltech Center for Environmental Microbial Interactions. M.T.B. was supported by an NSF GRFP fellowship. This research was supported by the National Institutes of Health (R01-EB018975 to M.G.S.) and Pew Charitable Trust. Related research in the Shapiro Laboratory is supported by the David and Lucille Packard Foundation, the Burroughs Wellcome Fund and the Heritage Medical Research Institute. Data and code availability: Plasmids will be made available through Addgene upon publication. All other materials and data are available from the corresponding author upon reasonable request. Author Contributions: R.C.H., M.T.B., K.W., M.B.S., P.D., Z.J., and R.D. planned and performed experiments. M.T.B. performed all in vivo experiments, with help from M.B.S. P.D. performed TEM imaging. D.R.M. built the ultrasound plate-scanning setup, and D.R.M. and D.P.S. wrote the associated MATLAB scripts for controlling it. Z.J. and D.P.S. wrote the MATLAB scripts for ultrasound imaging of EcN in vitro and in vivo. Z.J. performed the calibration of the L22-14v transducer. M.H.A. provided the axe-txe stability cassette, and advised on tumor colonization experiments. R.C.H., M.T.B., D.P.S., P.D., and Z.J. analyzed data. R.C.H., M.T.B., and M.G.S. wrote the manuscript with input from all other authors. M.G.S. supervised the research. The authors have declared no competing interest.
Group:Heritage Medical Research Institute
Funding AgencyGrant Number
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
Pew Charitable TrustUNSPECIFIED
David and Lucile Packard FoundationUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Heritage Medical Research InstituteUNSPECIFIED
Record Number:CaltechAUTHORS:20210429-092722876
Persistent URL:
Official Citation:Genomically Mined Acoustic Reporter Genes Enable On-Demand In Vivo Monitoring of Tumor-Homing Bacteria. Robert C. Hurt, Marjorie T. Buss, Katie Wong, Daniel P. Sawyer, Margaret B. Swift, Przemysław Dutka, David R. Mittelstein, Zhiyang Jin, Mohamad H. Abedi, Ramya Deshpande, Mikhail G. Shapiro. bioRxiv 2021.04.26.441537; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108863
Deposited By: Tony Diaz
Deposited On:29 Apr 2021 18:27
Last Modified:29 Apr 2021 18:27

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