A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants

Eva Yus, Jae-Seong Yang, Adrià Sogues and Luis Serrano ()
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Eva Yus: The Barcelona Institute for Science and Technology
Jae-Seong Yang: The Barcelona Institute for Science and Technology
Adrià Sogues: The Barcelona Institute for Science and Technology
Luis Serrano: The Barcelona Institute for Science and Technology

Nature Communications, 2017, vol. 8, issue 1, 1-12

Abstract: Abstract Quantitative analysis of the sequence determinants of transcription and translation regulation is relevant for systems and synthetic biology. To identify these determinants, researchers have developed different methods of screening random libraries using fluorescent reporters or antibiotic resistance genes. Here, we have implemented a generic approach called ELM-seq (expression level monitoring by DNA methylation) that overcomes the technical limitations of such classic reporters. ELM-seq uses DamID (Escherichia coli DNA adenine methylase as a reporter coupled with methylation-sensitive restriction enzyme digestion and high-throughput sequencing) to enable in vivo quantitative analyses of upstream regulatory sequences. Using the genome-reduced bacterium Mycoplasma pneumoniae, we show that ELM-seq has a large dynamic range and causes minimal toxicity. We use ELM-seq to determine key sequences (known and putatively novel) of promoter and untranslated regions that influence transcription and translation efficiency. Applying ELM-seq to other organisms will help us to further understand gene expression and guide synthetic biology.

Date: 2017
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DOI: 10.1038/s41467-017-00239-7

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