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Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries
The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify prot...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633160/ https://www.ncbi.nlm.nih.gov/pubmed/26536118 http://dx.doi.org/10.1371/journal.pone.0140730 |
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author | Stowe, Cassandra Pizzey, Arnold Kalber, Tammy Badar, Adam Lythgoe, Mark Pule, Martin |
author_facet | Stowe, Cassandra Pizzey, Arnold Kalber, Tammy Badar, Adam Lythgoe, Mark Pule, Martin |
author_sort | Stowe, Cassandra |
collection | PubMed |
description | The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm(2). We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. |
format | Online Article Text |
id | pubmed-4633160 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-46331602015-11-13 Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries Stowe, Cassandra Pizzey, Arnold Kalber, Tammy Badar, Adam Lythgoe, Mark Pule, Martin PLoS One Research Article The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm(2). We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. Public Library of Science 2015-11-04 /pmc/articles/PMC4633160/ /pubmed/26536118 http://dx.doi.org/10.1371/journal.pone.0140730 Text en © 2015 Stowe et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Stowe, Cassandra Pizzey, Arnold Kalber, Tammy Badar, Adam Lythgoe, Mark Pule, Martin Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries |
title | Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries |
title_full | Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries |
title_fullStr | Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries |
title_full_unstemmed | Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries |
title_short | Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries |
title_sort | flow-based single cell deposition for high-throughput screening of protein libraries |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633160/ https://www.ncbi.nlm.nih.gov/pubmed/26536118 http://dx.doi.org/10.1371/journal.pone.0140730 |
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