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Title: Engineering the S-Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials

Abstract

Materials synthesized by organisms, such as bones and wood, combine the ability to self-repair with remarkable mechanical properties. This multifunctionality arises from the presence of living cells within the material and hierarchical assembly of different components across nanometer to micron scales. While creating engineered analogs of these natural materials is of growing interest, our ability to hierarchically order materials using living cells largely relies on engineered 1D protein filaments. Here, we lay the foundations for bottom-up assembly of engineered living material composites in 2D along the cell body using a synthetic biology approach. We engineer the paracrystalline surface-layer (S-layer) of Caulobacter crescentus to display SpyTag peptides that form irreversible isopeptide bonds to SpyCatcher-modified proteins, nanocrystals, and biopolymers on the extracellular surface. Using flow cytometry and confocal microscopy, we show that attachment of these materials to the cell surface is uniform, specific, and covalent, and its density can be controlled based on the location of the insertion within the S-layer protein, RsaA. Furthermore, we leverage the irreversible nature of this attachment to demonstrate via SDS-PAGE that the engineered S-layer can display a high density of materials, reaching 1 attachment site per 288 nm2. Lastly, we show that ligation of quantum dotsmore » to the cell surface does not impair cell viability and this composite material remains intact over a period of two weeks. Taken together, this work provides a platform for self-organization of soft and hard nanomaterials on a cell surface with precise control over 2D density, composition, and stability of the resulting composite, and is a key step towards building hierarchically-ordered engineered living materials with emergent properties.« less

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [1]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); USDOE Advanced Research Projects Agency - Energy (ARPA-E); National Institutes of Health (NIH)
OSTI Identifier:
1542356
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; RsaA; engineered living materials; quantum dots; Caulobacter; biomaterial

Citation Formats

Charrier, Marimikel, Li, Dong, Mann, Victor R., Yun, Lisa, Jani, Sneha, Rad, Behzad, Cohen, Bruce E., Ashby, Paul D., Ryan, Kathleen R., and Ajo-Franklin, Caroline M. Engineering the S-Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials. United States: N. p., 2018. Web. doi:10.1021/acssynbio.8b00448.
Charrier, Marimikel, Li, Dong, Mann, Victor R., Yun, Lisa, Jani, Sneha, Rad, Behzad, Cohen, Bruce E., Ashby, Paul D., Ryan, Kathleen R., & Ajo-Franklin, Caroline M. Engineering the S-Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials. United States. doi:10.1021/acssynbio.8b00448.
Charrier, Marimikel, Li, Dong, Mann, Victor R., Yun, Lisa, Jani, Sneha, Rad, Behzad, Cohen, Bruce E., Ashby, Paul D., Ryan, Kathleen R., and Ajo-Franklin, Caroline M. Fri . "Engineering the S-Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials". United States. doi:10.1021/acssynbio.8b00448. https://www.osti.gov/servlets/purl/1542356.
@article{osti_1542356,
title = {Engineering the S-Layer of Caulobacter crescentus as a Foundation for Stable, High-Density, 2D Living Materials},
author = {Charrier, Marimikel and Li, Dong and Mann, Victor R. and Yun, Lisa and Jani, Sneha and Rad, Behzad and Cohen, Bruce E. and Ashby, Paul D. and Ryan, Kathleen R. and Ajo-Franklin, Caroline M.},
abstractNote = {Materials synthesized by organisms, such as bones and wood, combine the ability to self-repair with remarkable mechanical properties. This multifunctionality arises from the presence of living cells within the material and hierarchical assembly of different components across nanometer to micron scales. While creating engineered analogs of these natural materials is of growing interest, our ability to hierarchically order materials using living cells largely relies on engineered 1D protein filaments. Here, we lay the foundations for bottom-up assembly of engineered living material composites in 2D along the cell body using a synthetic biology approach. We engineer the paracrystalline surface-layer (S-layer) of Caulobacter crescentus to display SpyTag peptides that form irreversible isopeptide bonds to SpyCatcher-modified proteins, nanocrystals, and biopolymers on the extracellular surface. Using flow cytometry and confocal microscopy, we show that attachment of these materials to the cell surface is uniform, specific, and covalent, and its density can be controlled based on the location of the insertion within the S-layer protein, RsaA. Furthermore, we leverage the irreversible nature of this attachment to demonstrate via SDS-PAGE that the engineered S-layer can display a high density of materials, reaching 1 attachment site per 288 nm2. Lastly, we show that ligation of quantum dots to the cell surface does not impair cell viability and this composite material remains intact over a period of two weeks. Taken together, this work provides a platform for self-organization of soft and hard nanomaterials on a cell surface with precise control over 2D density, composition, and stability of the resulting composite, and is a key step towards building hierarchically-ordered engineered living materials with emergent properties.},
doi = {10.1021/acssynbio.8b00448},
journal = {ACS Synthetic Biology},
number = 1,
volume = 8,
place = {United States},
year = {2018},
month = {12}
}

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