A Molecular Device for Tuneable Evolution

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Evolution is a unique property of biological systems and a challenge for Engineering Biology. It canplay a double role: On one hand it can be undesirable, as evolution may lead to mutationsinactivating an engineered function. On the other hand, evolution creates variation which can leadto improving functions of interest or create new ones.In this project we will exploit this dual role to create a molecular tool that can operate in a cell toboth prevent mutations resulting in loss of function and generate diversity for directed evolutionon demand. This will be achieved by using retrons. Retrons are DNA elements that encode for areverse transcriptase and a single-stranded DNA/RNA hybrid (msDNA) 1 . Once the retron istranscribed, the reverse transcriptase generates the msDNA and a fragment of it gets integratedback into the lag strand of the bacterial chromosome by recombination.Retrons can be engineered and used to target a region of interest in the DNA of bacteria. Theyhave been used for DNA storage 2 , as they generate a record of cellular events in the form ofpermanent modifications to the DNA. Moreover, because the msDNA sequence can harbour up to80 bp of a cargo DNA, retrons can be used for direct mutagenesis of a target sequence 3 or to edit asite of interest by recombination. We have preliminary results demonstrating the in vivomutagenesis in Escherichia coli of the rpoB gene encoding the beta subunit of the RNApolymerase. Our results show that a wild-type rpoB can be replaced by a retron-borne segmentconferring resistance to the antibiotic rifampicin. Conversely, we have shown that mutations in thegene rpoB can be rescued with a retron carrying the wildtype sequence of the gene