Open AccessRationally designed Spot 42 RNAs with an inhibition/toxicity profile advantageous for engineering E. coli
Author(s) -
Vogel Carolin,
Gynnå Arvid H.,
Yuan Jinwen,
Bao Letian,
Liljeruhm Josefine,
Forster Anthony C.
Publication year - 2020
Publication title -
engineering reports
Language(s) - English
Resource type - Journals
ISSN - 2577-8196
DOI - 10.1002/eng2.12126
Subject(s) - escherichia coli , transfer rna , translation (biology) , biology , rna , gene , stop codon , antisense rna , codon usage bias , computational biology , genetics , messenger rna , genome
Bacterial regulatory small RNAs (sRNAs) have shown promise for gene knock‐down studies and metabolic engineering. However, some mRNAs might be difficult to target due to poor binding by the Hfq chaperone, individual synthetic sRNAs can have off‐target effects, potential sRNA toxicities have not been studied globally, and a consensus on optimal sRNA design has yet to emerge. Here, Spot 42 sRNA is validated as an excellent scaffold by showing that its over‐expression minimally affects the growth rate of Escherichia coli , and that inhibition is reliably achieved for all eight tested protein targets by designing antisense to target the first few codons. Two related sRNAs that could not be cloned, possibly due to lethality of the encoded sRNAs, became clonable when an eight‐nucleotide sequence was inserted directly upstream of the antisense region. Global fitness costs for E. coli of the designer sRNAs were measured and found to be variable but tolerable. Importantly for utility, there was no correlation between target inhibition and cellular toxicity. As a proof of concept for applications, suppression of the UAG stop codon was improved by knock down of translation release factor 1 (RF1).