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CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion.
Metab Eng
January 2023
MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China; Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China. Electronic address:
Genome-scale target identification promises to guide microbial cell factory engineering for higher-titer production of biomolecules such as recombinant proteins (r-protein), but challenges remain due to the need not only for comprehensive genotypic perturbation but also in conjunction with high-throughput phenotypic screening strategies. Here, we developed a CRISPRi-microfluidics screening platform to systematically identify crucial gene targets that can be engineered to enhance r-protein secretion in Corynebacterium glutamicum. We created a CRISPR interference (CRISPRi) library containing 46,549 single-guide RNAs, where we aimed to unbiasedly target all genes for repression.
View Article and Find Full Text PDFA FlAsH-tetracysteine assay for quantifying the association and orientation of transmembrane α-helices.
Chembiochem
May 2011
Department of Chemistry, Boston College, Chestnut Hill, MA 02466, USA.
Protein labeling with FlAsH and ReAsH.
Methods Mol Biol
November 2006
Invitrogen Corporation, Madison, WI, USA.
The ability to image biochemical and phenotypical changes in living cells has become crucial for the investigation and understanding of the molecular mechanisms that govern all physiological cellular functions in health and disease. Genetically encoded reporters derived from fluorescent proteins (FPs) have proved to be extremely useful for localization and interaction studies in living cells. However, the large size and spectral properties of FP impose certain limitations for their use.
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