Project description:Quantitative analysis of the sequence determinants of transcription and translation regulation is of special relevance for systems and synthetic biology applications. Here, we developed a novel generic approach for the fast and efficient analysis of these determinants in vivo. ELM-seq (expression level monitoring by DNA methylation) uses Dam coupled to high-throughput sequencing) as a reporter that can be detected by DNA-seq. We used the genome-reduced bacterium Mycoplasma pneumoniae to show that it is a quantitative reporter. We showed that the methylase activity correlates with protein expression, does not affect cell viability, and has a large dynamic range (~10,000-fold). We applied ELM-seq to randomized libraries of promoters or 5’ untranslated regions. We found that transcription is greatly influenced by the bases around the +1 of the transcript and the Pribnow box, and we also identified several epistatic interactions (including the +1 and the “extended Pribnow”). Regarding translation initiation, we confirmed that the Shine-Dalgarno motif is not relevant, but instead, that RNA secondary structure is the main governing factor. With this in hand, we developed a predictor to help tailor gene expression in M. pneumoniae. The simple ELM-seq methodology will allow identifying and optimizing key sequence determinants for promoter strength and translation. The ELM-seq methodology allows both researchers and companies to identify and optimize in an easy and comprehensive manner, key sequence determinants for promoter strength and translation.
Project description:We compared 10, 20 and 40 animal pools to determine the best number of intact adults to pool Keywords: Agilent microarray, pooling, Drosophila, intact adults Overall design: 16 vials of flies were reared. From each vial 5 individuals were selected. For the 10 animal comparison 4 vials were used with each replicate being a pool of 5 animals over 2 independent vials. For the 20 animal comparison 8 vials were used with each replicate being a pool of 5 animals over 5 independent vials and for the 40 animal comparison each replicate was a pool of 5 animals over 8 independent vials. There are two independent replicates of each type for 6 samples which were hybridized on three chips in a loop design.
Project description:To dissect local regulation into cis and trans contribution, we measured allelic differential expression in a hybrid cross of two yeast strains and compared it against allelic differential expression in a pool of spores of the same cross. RNA- and DNA-seq of a diploid S. cerevisiae hybrid and its haploid spores.