Expression level monitoring by DNA methylation and sequencing (ELM-seq) allows optimizing sequence determinants of gene expression and translation comprehensively and more reliably than classical methods.

The method can be of special relevance for bioengineering and industrial biotechnology, for the biosynthesis of useful molecules such as drugs, enzymes, antibiotics, organic acids, biofuels and bioplastics.

Technology:

Quantitative determination of the key sequences that influence transcription and translation efficiency (promoters and other regulatory sequences) is of special relevance for systems and synthetic biology, in order to understand, and eventually adjust the level of expression of a certain gene or set of genes (bioingeneering). It can also be relevant to optimize protein production in many different contexts or applications.  Screening methodologies that combine the expression of fluorescent proteins or antibiotic resistance markers under the control of randomized sequences show relatively low sensitivity and dynamic range, while the use of antibiotic resistance may alter the viability of clones and lead to false positives. Other sorting techniques, like FACs are laborious and low reproducible, and can be limited by several factors (e.g. pH, cell aggregation and cell shape and size, particularly in bacteria), also leading to bias.  ELM-seq overcomes these limitations. It uses E. coli DNA adenine methylase (DAM) as a reporter coupled with methylation-sensitive (DpnI) vs unsensitive (MboI) restriction enzyme digestion and PCR to enable the quantitative analyses of the effect of known or novel regulatory sequences on the efficiency of transcription and translation in vivo (DAMRatio). Importantly, the method can also help distinguish from soluble and insoluble (aggregated) proteins.

Advantages:

• Robust – based on PCR
• Universal – DAM works well in many bacteria and eukaryotic systems
• Quantitative – DAMRatio reflects protein levels over a very wide dynamic range (~10,000-fold in the system tested)
• Unbiased – it does not need cell sorting nor selection (e.g. antibiotic)
• Amenable to high throughput – it can be used with random libraries coupled with high-throughput sequencing for a comprehensive analysis of determinants of gene expression and translation in vivo
• It can be used to analyze multiple proteins in parallel (“multiplexing”)
• It can be used to test protein solubility in high throughput

References:

Yus et al. (2017) A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants. Nature Communications 8: 368

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