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.
Highlights
The identification and isolation of proteins having refined or novel characteristics is an important area of research in many scientific fields, examples include therapeutics[1], antibody production[2] and various imaging modalities[3,4]
Hoechst 34580 fluorescence was selected as the primary sort determinate; and further electronic gates were applied to the data, selecting for bacteria having light scatter properties and Hoechst 34580 staining which correlate with viable cells (Fig 1b)
In contrast to standard bulk sorting modalities, we adopted a single-cell approach where single bacterial cells meeting all relevant sort criteria were deposited in a dense matrix onto the surface of solid bacterial growth medium (Fig 1c)
Summary
The identification and isolation of proteins having refined or novel characteristics is an important area of research in many scientific fields, examples include therapeutics[1], antibody production[2] and various imaging modalities[3,4]. Proteins used in imaging technologies such as bioluminescent proteins[11,12] for optical imaging or complex fluorescent proteins[13,14] for super-high resolution microscopy (PALM[15]/STORM[16]) are difficult to screen in a high-throughput manner In these cases, the requirement for single cell analysis means liquid handling robots are of limited facility so current screening methods are largely restricted to traditional, microbiological techniques which are poorly matched with the more recent advances in genetic biology, which typically produce very large libraries of mutant proteins[17,18]. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto solid nutrient growth media in a dense matrix format We show this matrix format is readily applicable to high-throughput machine interrogation and interpretation, enabling the large scale screening of the protein library. To further enhance this screening platform, we have developed an electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the speed of colony deposition by almost 10-fold
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