Project description:We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC, which was achieved within 15 min from sample injection to fraction collection. Ribo Mega-SEC reproducibly shows translating ribosomes exist predominantly in polysome complexes in extracts isolated from human cell lines and mouse liver tissue, which alter in response to starvation. Ribo Mega-SEC provides a rapid, efficient, convenient and highly reproducible method for studying functional translation complexes and is easily combined with high-through put analysis such as proteomics and RNA-Seq, or with structural analysis using electron microscopy. We propose that Ribo Mega-SEC analysis is an accessible alternative to traditional polysome profiling using sucrose density gradients.
Project description:CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR/Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed MEGA (Multiplexed Effector Guide Arrays), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR/Cas13d. MEGA enables quantitative, reversible, and massively-multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
Project description:Transcriptional profiling of P. putida KT2440 cells comparing untreated cells with 1 mM indole or 50 μg/ml ampicillin or 1 mM indole plus 50 μg/ml ampicillin treated cells
Project description:Transcriptional profiling of A. oleivorans DR1 treated Ampicillin 100 M-BM-5g/ml for 15min . To identify effect of ampicillin in A. oleivornas DR1, the cells were grown to exponential phase (OD600 ~0.4) and treated ampicillin 100 M-BM-5g/ml for 15 min.
Project description:Transcriptional profile of Pseudomonas putida DOT-T1E grown in the presence of ampicillin (300 ug/ml) compared with the same cells grown in absence of ampicillin
Project description:Transcriptional profile of Pseudomonas putida DOT-T1E-18 grown in the presence of ampicillin (100 ug/ml) compared with the same cells grown in absence of ampicillin.