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:Adenosine-to-inosine A-to-I mRNA editing alters genetic information post-transcriptionally and can impact protein sequence and function, yet its regulation in bacteria remains unclear. Here, we profiled A-to-I editing in Escherichia coli across nutrient-rich LB and minimal M9 media and different growth phases. Our analysis expanded the repertoire of TadA-dependent A-to-I edited mRNAs to 27, including 12 novel sites, and revealed that editing levels were dynamic and markedly increased at stationary phase in LB but not in M9. Editing levels were independent of mRNA expression yet correlated with tRNA-Arg2 downregulation, and overexpressing tRNA-Arg2 reduced mRNA editing, demonstrating substrate competition for TadA, the sole bacterial tRNA adenosine deaminase. Mutants with TadA-deficient editing or reduced tRNA-Arg2 expression displayed similar LB-specific growth defects. Moreover, tRNA-Arg2 expression, tRNA-Arg2-dependent codon usage, and tRNA-Arg2 editing were all elevated in LB compared to M9. These findings establish regulatory principles for bacterial RNA editing, implicate tRNA editing in nutrient-responsive fitness, and provide a framework to explore the physiological roles of mRNA editing
Project description:Ultra-sensitive quantification of heterogeneous mutations reveals the replication-directed genomic asymmetry in Escherichia coli generated through accelerated laboratory evolution
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: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.
