Project description:It is known that current the-art-of-the-state TadA8 and TadA8e which evolved from E. coli TadA. They inherited the 'YA' context from tRNA deaminase. We started with wildtype E. coli TadA and designed an evolution campaign to force TadA variants to deaminate “GA” with fast kinetics. Three rounds of de novo directed evolution followed by DNA shuffling led to TadA8r, a TadA variant of superior “RA” deamination activity. TadA8r acts on a broadened editing window when fused to Streptococcus pyogenes Cas9 (SpCas9) and delivers robust editing at PAM distal positions. While highly active at on-target sites, ABE8r shows off-target DNA and RNA editing much lower than ABE8e. The off-target effects of ABE8r can be further mitigated by introducing a V106W substitution23, a R153 deletion22, or by mRNA delivery. Lastly, we demonstrate ABE8r-mediated correction of G1961E in ABCA4, the most prevalent mutation driving Stargardt disease (STGD1), in a “GA” context. ABE8r, with its superior activity and broadened context compatibility, complements and expands the current ABE family.
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:Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. ABEs function most effectively when the target A is in a TA context. Deficient ABE processing of RA (R = A or G) is most evident when the target A is outside the comfortable editing window or when delivery is suboptimal. In the current study, we report directed evolution of TadA8r, a new variant of the Escherichia coli tRNA-specific adenosine deaminase (TadA) with ultra-fast deoxyadenosine deamination and no context bias.
Project description:To understand the mechanism of isopropanol tolerance of Escherichia coli for improvement of isopropanol production, we performed genome re-sequencing and transcriptome analysis of isopropanol tolerant E. coli strains obtained from parallel adaptive laboratory evolution under IPA stress.
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:Ultra-sensitive quantification of heterogeneous mutations reveals the replication-directed genomic asymmetry in Escherichia coli generated through accelerated laboratory evolution
Project description:Laboratory adaptive evolution experiments were conducted using serial passage of E. coli in M9 minimal medium supplemented with either 2 g/L of lactate for 60 days or 2 g/L of glycerol for 44 days. 7 parallel evolution strains were generated for growth on lactate and 7 parallel evolution strains were generated for growth on glycerol. Affymetrix arrays were used to study the time-course change in gene expression from unevolved E. coli (day 0) to a midpoint evolved strain (day 20) and evolutionary endpoints
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:Avian pathogenic Escherichia coli strains frequently cause extra-intestinal infections and are responsible for significant economic losses in the poultry industry worldwide. APEC isolates are closely related to human extraintestinal pathogenic E.coli strains and may also act as pathogens for humans. In this work, three type VI secretion systems were deleted to analyze which pathogenicity characteristics would change in the mutants, compared to wild type strain (SEPT 362).
