title: Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.
abstract: 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/cm2. 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.
authors.affiliation: Cancer Institute, Department of Haematology, Division of Medicine, University College London, London, United Kingdom.|Centre for Advanced Biomedical Imaging, Division of Medicine, University College London, London, United Kingdom.
authors.forename: Cassandra
authors.initials: C
authors.lastname: Stowe
authors.affiliation: Cancer Institute, Department of Haematology, Division of Medicine, University College London, London, United Kingdom.
authors.forename: Arnold
authors.initials: A
authors.lastname: Pizzey
authors.affiliation: Centre for Advanced Biomedical Imaging, Division of Medicine, University College London, London, United Kingdom.
authors.forename: Tammy
authors.initials: T
authors.lastname: Kalber
authors.affiliation: Centre for Advanced Biomedical Imaging, Division of Medicine, University College London, London, United Kingdom.
authors.forename: Adam
authors.initials: A
authors.lastname: Badar
authors.affiliation: Centre for Advanced Biomedical Imaging, Division of Medicine, University College London, London, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Lythgoe
authors.affiliation: Cancer Institute, Department of Haematology, Division of Medicine, University College London, London, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: Pule
mesh_terms: D001419:Bacteria / Q000302:isolation & purification / Q000378:metabolism*; D019295:Computational Biology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D010843:Pichia / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011506:Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins