Project description:These E. coli strains were grown with various signaling molecules and the expression profiles were determined. Keywords: addition of quorum and host hormone signals
Project description:Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.
Project description:Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and – for AcrIIA5 – indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.
2024-03-01 | GSE254101 | GEO
Project description:VIM-2 deep mutational scanning
Project description:Comparison of the whole genome gene expression level of an amoxicillin resistant E. coli strain with the wildtype it was derived from. The process of amoxicillin adaptation of E. coli MG1655 wildtype cells is further descibed in van der Horst, M, J.M. Schuurmans, M. C. Smid, B. B. Koenders, and B. H. ter Kuile (2011) in Microb. Drug Resist. 17:141-147. Resistance to amoxicillin was induced in E. coli by growth in the presence of stepwise increasing antibiotic concentrations. To investigate consequences of the aquisition of amoxicillin resistance the transcriptomic profile of sensitive and resistant cells was compared in the absence and presence of sub-inhibitory (0.25xMIC) amoxicillin concentrations was compared.
Project description:In order to understand the impact of genetic variants on transcription and ultimately in changes in observed phenotypes we have measured transcript levels in an Escherichia coli strains collection, for which genetic and phenotypic data has also been measured.
Project description:These E. coli strains were grown with various signaling molecules and the expression profiles were determined. Experiment Overall Design: These E. coli strains were grown with various signaling molecules and the expression profiles were determined using the Affymetrix E. coli 2.0 Array. Experiment Overall Design: Comparisons of the profiles were used to guide further examination using RT-PCR.
Project description:The purpose of this study is to determine whether the presence of pathogenic Escherichia coli in colon is associated with psychiatric disorders.
