1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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2. |
- Gong, Guiping, et al.
(författare)
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GTR 2.0: GRNA-tRNA Array and Cas9-NG Based Genome Disruption and Single-Nucleotide Conversion in Saccharomyces cerevisiae
- 2021
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:6, s. 1328-1337
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Tidskriftsartikel (refereegranskat)abstract
- Targeted genome disruptions and single-nucleotide conversions with the CRISPR/Cas system have greatly facilitated the development of gene therapy, basic biological research, and synthetic biology. With vast progress in this field, there are still aspects to be optimized, including the target range, the ability to multiplex, the mutation efficiency and specificity, as well as the requirement of adjusting protospacer adjacent motifs (PAMs). Here, we report the development of a highly efficient genome disruption and single-nucleotide conversion tool with a gRNA-tRNA array and SpCas9-NG (GTR 2.0). We performed gene disruptions in yeast cells covering all 16 possible NGN PAMs and all 12 possible single-nucleotide conversions (N to N) with near 100% efficiencies. Moreover, we applied GTR 2.0 for multiplexed single-nucleotide conversions, resulting in 66.67% mutation efficiency in simultaneous generation of 4 single-nucleotide conversions in one gene, as well as 100% mutation efficiency for simultaneously generating 2 single-nucleotide conversions in two different genes. GTR 2.0 will substantially expand the scope, efficiency, and capabilities of yeast genome editing, and will be a versatile and invaluable addition to the toolbox of synthetic biology and metabolic engineering.
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