Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology
| Autor: | Jose Duarte, Yolanda Schaerli, Içvara Barbier |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
High-throughput screening Microfluidics Biomedical Engineering General Medicine Computational biology Cells Immobilized Biology Directed evolution Biochemistry Genetics and Molecular Biology (miscellaneous) Molecular biology 03 medical and health sciences Synthetic biology Fluorescence-Activated Cell Sorting chemistry.chemical_compound 030104 developmental biology chemistry cell-to-cell variability combinatorial libraries directed evolution hydrogel beads screening synthetic biology Escherichia coli Screening method Agarose Synthetic Biology Synthetic construct Gene Library |
| Zdroj: | ACS synthetic biology, vol. 6, no. 11, pp. 1988-1995 |
| Popis: | Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states. |
| Databáze: | OpenAIRE |
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