[ { "id": "authors:mtg7y-qfj83", "collection": "authors", "collection_id": "mtg7y-qfj83", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211216-235830370", "type": "conference_item", "title": "Directed evolution of a genetically encoded contrast agent for ultrasound", "author": [ { "family_name": "Hurt", "given_name": "Robert C.", "orcid": "0000-0002-4347-6901", "clpid": "Hurt-Robert-C" }, { "family_name": "Wong", "given_name": "Katie", "clpid": "Wong-Katie" }, { "family_name": "Sawyer", "given_name": "Daniel", "orcid": "0000-0003-2926-191X", "clpid": "Sawyer-Daniel-P" }, { "family_name": "Deshpande", "given_name": "Ramya", "clpid": "Deshpande-Ramya" }, { "family_name": "Mittelstein", "given_name": "David R.", "orcid": "0000-0001-8747-0483", "clpid": "Mittelstein-David-R" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "abstract": "A major challenge in the field of biol. imaging and synthetic biol. is noninvasively visualizing the function of natural and engineered cells inside opaque samples such as living animals. One promising technol. that addresses this limitation is ultrasound, with its penetration depth of several cm and spatial resoln. of tens of mm. Recently, the first genetically encoded ultrasound contrast agents-gas vesicles (GVs)-were developed to link ultrasound to mol. and cellular function via heterologous expression in both commensal bacteria and mammalian cells. GVs are air-filled protein nanostructures derived from buoyant photosynthetic microbes, in which they serve as cellular flotation devices. GVs are encoded by operons of 8-14 genes, and most of their mol. makeup comprises the structural protein GvpA. The air inside GVs allows them to scatter ultrasound. Just as the discovery of the first fluorescent proteins was followed by the engineering and evolution of their properties, we are working to engineer the properties of GVs as acoustic reporters. Here, we pursue this goal using directed evolution by both devising a strategy for high-throughput acoustic screening of GVs in bacterial colonies and validating its ability to identify new phenotypes in mutant libraries. We generated scanning site satn. libraries for two homologs of GvpA and screened them in E. coli using a custom-built robotic ultrasound plate scanner. Custom imaging pulse sequences were used to assess the acoustic phenotypes of each colony, including total backscattering, nonlinear scattering, and collapse pressure. Using this technique, we identified mutants of GvpA with >150x higher acoustic signal than their parents. These techniques will enable directed evolution to play as big a role in the engineering of acoustic biomols. as it has in the development of their fluorescent counterparts.", "publisher": "Caltech Library", "publication_date": "2021-08" }, { "id": "authors:tjy1r-6b253", "collection": "authors", "collection_id": "tjy1r-6b253", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211216-234818185", "type": "conference_item", "title": "Toward a thermally self-regulating living material", "author": [ { "family_name": "Xiong", "given_name": "Lealia", "clpid": "Xiong-Lealia" }, { "family_name": "Garrett", "given_name": "Michael A.", "clpid": "Garrett-Michael-A" }, { "family_name": "Kornfield", "given_name": "Julia Ann", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "abstract": "Engineered living materials (ELMs) retain desirable characteristics of the living component, such as exponential growth, self-repair, and responsiveness to external stimuli. Escherichia coli are a promising constituent of ELMs because they are very tractable to genetic engineering.Variation in ambient temp. presents a challenge in deploying ELMs outside of a lab. environment. E. coli experience maximal growth near 37\u00b0C. In addn., E. coli protein synthesis decreases below 37\u00b0C, while protein misfolding and aggregation tends to increase with temp.Here, we develop a genetically encoded\nmechanism for autonomous temp. homeostasis in ELMs contg. E. coli by engineering circuits that change expression of a light-absorptive chromophore in response to changes in temp. Our simulations show that by increasing absorptivity below 36\u00b0C, the material will heat above the ambient temp. to preserve optimal growth and protein expression, and thus material functionality.We program bacteria to respond to temp. using temp.-sensitive transcriptional repressors (TSRs). \n\nTwo families of TSRs with switching temps. ranging from 36\u00b0C to 44\u00b0C have been developed in our lab by directed evolution of TcI, a temp.-sensitive mutant of bacteriophage l repressor cI, and TlpA, a transcriptional auto-repressor from the virulence plasmid of Salmonella typhimurium. These thermal bioswitches can be further tuned for optimal switching in the ELM application. Formation of a black chromophore from a pale yellow precursor is enzymically catalyzed. Integrating the gene for this enzyme into a genetic circuit with a down-shifted mutant of TlpA enables E. coli to express black chromophore at low temps. and not at high temps.We measure the ability of patches of E. coli (simulating an E. coli-based ELM) to grow in a custom lighted incubator at different ambient temps. by observing patch diam. and thickness. We continuously monitor E. coli temp. under illumination using thermal IR imaging with custom controller software. Comparison of exptl. results and simulations will be presented. We demonstrate thermal control of pigmentation and resulting increase in sample temp.", "publisher": "Caltech Library", "publication_date": "2021-08" }, { "id": "authors:ggdpr-s7245", "collection": "authors", "collection_id": "ggdpr-s7245", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181101-133603645", "type": "conference_item", "title": "Genetically encoded acousto-magnetic protein nanostructures for non-invasive imaging of cellular functions", "author": [ { "family_name": "Lu", "given_name": "George", "orcid": "0000-0002-4689-9686", "clpid": "Lu-George-Jiaozhi" }, { "family_name": "Farhadi", "given_name": "Arash", "orcid": "0000-0001-9137-8559", "clpid": "Farhadi-A" }, { "family_name": "Szablowski", "given_name": "Jerzy", "orcid": "0000-0001-7851-5408", "clpid": "Szablowski-J-O" }, { "family_name": "Lee-Gosselin", "given_name": "Audrey", "orcid": "0000-0002-2431-2741", "clpid": "Lee-Gosselin-A" }, { "family_name": "Barnes", "given_name": "Samuel", "orcid": "0000-0002-1065-8442", "clpid": "Barnes-S-R" }, { "family_name": "Lakshmanan", "given_name": "Anupama", "orcid": "0000-0002-6702-837X", "clpid": "Lakshmanan-A" }, { "family_name": "Bourdeau", "given_name": "Raymond", "orcid": "0000-0003-2202-1980", "clpid": "Bourdeau-R-W" }, { "family_name": "Shapiro", "given_name": "Mikhail", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "abstract": "Genetically encoded optical reporters such as green fluorescent protein (GFP) have revolutionized biomedical research\nby enabling observations of specific biol. processes in engineered cells and transgenic animals. However, such optical\nagents are fundamentally limited by the ~ 1 mm penetration depth of light in opaque tissues. As cellular therapies\nadvance towards rodent models and ultimately humans, this limitation becomes increasingly severe. Therefore, we aim\nto develop new classes of reporter genes for non-invasive imaging modalities that utilize deeply penetrant forms of\nenergy, such as magnetic fields and sound waves. Here, we describe the first reporter genes for acoustically modulated\nmagnetic resonance imaging (AM-MRI), a modality that combines MRI and ultrasound. These agents are based on gas\nvesicles (GVs), a class of gas-filled protein nanostructures evolved in photosynthetic microbes as a means to regulate\ntheir buoyancy. GVs are hundreds of nanometers in size, and transferring the gene cluster into E. coli enables their\nformation inside these bacteria. The air inside GVs allows them to be detected at nanomolar concns. by susceptibilitybased\nMRI. Uniquely, such contrast is \"erasable\" by ultrasound pulses at specific pressures, which permits selective\nimaging of these agents without background tissue contrast that has plagued the use of existing MRI contrast agents.\nFurthermore, gene orthologs encode GVs of different size, shape and ultrasound-responsive pressure, which in turn give\nrise to differential MRI contrast and \"erasable\" pressure thresholds. Thus, multiplexed imaging can be achieved by\ngenetically encoding several types of GVs. Finally, the clustering of GVs induces a 10-fold enhancement of T2* contrast,\nwhich enables the potential design of sensors to dynamically report biol. signals. The ability of GVs to be genetically\nencoded and engineered opens the possibility of using this new form of imaging contrast in a wide range of applications,\nesp. in diagnosis and cellular therapeutics.", "publisher": "Caltech Library", "publication_date": "2018-08" }, { "id": "authors:2yv5z-1ax59", "collection": "authors", "collection_id": "2yv5z-1ax59", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160913-083410495", "type": "conference_item", "title": "Genetically encoded nanostructures for non-invasive imaging of biological systems", "author": [ { "family_name": "Shapiro", "given_name": "Mikhail", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "abstract": "Many important biological processes \u2013 ranging from simple metabolism to complex cognition \u2013 take place deep inside living organisms, yet our ability to study them in this context is limited. Technologies such as fluorescent and luminescent proteins enable exquisitely precise imaging of genetically specific cellular function in small and translucent specimens, but are limited by the poor penetration of light into larger tissues. In contrast, most non-invasive technologies such as magnetic resonance imaging (MRI) and ultrasound \u2013 while based on energy forms that penetrate tissue effectively \u2013 lack the needed molecular precision. Our work attempts to bridge this gap by engineering new molecular technologies that connect penetrant energy to specific aspects of cellular function in vivo. Here, I will describe molecular reporters for non-invasive imaging using MRI and ultrasound developed based on a unique class of genetically encoded gas-filled nanostructures called gas vesicles (GVs). These nanostructures evolved in photosynthetic microbes as a means to regulate buoyancy, and comprise a thin self-assembled protein shell enclosing a hollow interior with dimensions of approximately 250 nm. We have shown that the unique properties of GVs enable them to serve as sensitive molecular reporters in ultrasound, hyperpolarized ^(129)Xe MRI and susceptibility weighted MRI, representing the first genetically encodable reporters for each of these modalities. Furthermore, by engineering GVs at the genetic level, we can modify their contrast properties, mechanics and surface functionalization to enable new modes of imaging. In addition, by adapting GV-encoding gene clusters for expression in heterologous hosts, we are now able to use GVs as reporter genes to image engineered cells in vivo.", "publisher": "Caltech Library", "publication_date": "2016-08" } ]