Gene regulation in Escherichia coli is commonly selected for both high plasticity and low noise.

Bacteria often respond to dynamically changing environments by regulating gene expression. Despite this regulation being critically important for growth and survival, little is known about how selection shapes gene regulation in natural populations. To better understand the role natural selection plays in shaping bacterial gene regulation, here we compare differences in the regulatory behaviour of naturally segregating promoter variants from Escherichia coli (which have been subject to natural selection) to randomly mutated promoter variants (which have never been exposed to natural selection). We quantify gene expression phenotypes (expression level, plasticity and noise) for hundreds of promoter variants across multiple environments and show that segregating promoter variants are enriched for mutations with minimal effects on expression level. In many promoters, we infer that there is strong selection to maintain high levels of plasticity, and direct selection to decrease or increase cell-to-cell variability in expression. Taken together, these results expand our knowledge of how gene regulation is affected by natural selection and highlight the power of comparing naturally segregating polymorphisms to de novo random mutations to quantify the action of selection.

Efficiency of the synthetic self-splicing RiboJ ribozyme is robust to cis- and trans-changes in genetic background.

The expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between cis- or trans-genetic elements affect the behavior of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context-dependent cis-interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self-splicing ribozyme called RiboJ, which can be used to decouple upstream 5' UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans- and cis-genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis-effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis- nor trans-genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis- and trans-genetic changes.

Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding.

Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.