Project description:We explored functional redundancy of three structurally related KCTD proteins, KCTD2, KCTD5 and KCTD17 by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gb1 was involved in transcriptional control of gene expression, so we compared gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 lead to numerous changes in the expression levels of other G protein subunit genes while knockout of KCTD isoforms, had the opposite effect, presumably because of their role in regulating levels of Gb1. Our work demonstrates a unique relationship between KCTD proteins and Gb1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate.
Project description:An Adeno-Associated Virus capsid fitness landscape reveals a frameshifted viral gene and in vivo design principles, enabling machine-guided engineering.
2019-11-29 | GSE139657 | GEO
Project description:Ribo-seq guided design of enhanced protein secretion in Komagataella phaffii
Project description:The global escalation of antibiotic-resistant bacterial infections poses a life-threatening challenge to public health, necessitating the urgent development of innovative antibiotics targeting unexploited metabolic vulnerabilities. Through rational design, we developed a series of benzo-dioxygenated FabH inhibitors targeting the bacterial fatty acid biosynthesis pathway. Guided by active-site analysis and pharmacophore-guided optimization, we engineered a Y-shaped scaffold that achieved nanomolar inhibition of FabH (IC50 = 1.90 µM). The lead compound F35 showed broad-spectrum efficacy with MIC values as low as 1.56 µg/mL against Gram-negative and Gram-positive pathogens, outperforming Kanamycin B. Structural analysis revealed key interactions between FabH conserved residues and fluorine-mediated halogen bonding. In vivo assay, F35 accelerated wound closure in S. aureus-infected rodents, demonstrating a favorable biocompatibility. Our study establishes a convergence paradigm that integrates structure design, chemoproteomic identification, and therapeutic development for antibiotics, providing a strategic blueprint to combat multidrug resistance via precision targeting of metabolic chokepoints in bacterial pathogens.
Project description:Promoters play a central role in controlling gene regulation; however, a small set of promoters is used for most genetic construct design in the yeast Saccharomyces cerevisiae. The ability to generate and utilize models that accurately predict protein expression from promoter sequence may enable rapid generation of novel useful promoters, facilitating synthetic biology efforts in this model organism. We measured the activity of over 675,000 unique sequences in a constitutive promoter library, and over 327,000 sequences in a library of inducible promoters. Training an ensemble of convolutional neural networks jointly on the two datasets enabled very high (R2 > 0.79) predictive accuracies on multiple prediction tasks. We developed model-guided design strategies which yielded large, sequence-diverse sets of novel promoters exhibiting activities similar to current best-in-class sequences. In addition to providing large sets of new promoters, our results show the value of model-guided design as an approach for generating DNA parts.