A Molecular Device for Tuneable Evolution

进化是生物系统的独特属性，也是工程生物学面临的挑战。它可以扮演双重角色：一方面，它可能是不受欢迎的，因为进化可能会导致突变，从而激活工程功能。在这个项目中，我们将利用这一双重角色来创造一种分子工具，它可以在细胞中操作，既可以防止导致功能丧失的突变，又可以根据需要产生定向进化的多样性。这将通过使用回溯来实现。复制子是编码反向转录酶和单链DNA/RNA杂交体(MsDNA)的DNA元件。一旦逆转录酶被翻译，逆转录酶就会产生msDNA，它的一段片段通过重组被整合回细菌染色体的滞后链上。可以对反转录片段进行改造，并将其用于靶向细菌DNA中的感兴趣区域。它们被用于DNA存储，因为它们以DNA永久修改的形式产生细胞事件的记录。此外，由于msDNA序列可以包含高达80bp的货运DNA，反子可用于直接突变目标序列3或通过重组编辑感兴趣的Asite。我们已经有了初步的结果，证明了编码RNA聚合酶的β亚基的rpoB基因在大肠杆菌中能够活体突变。我们的结果表明，野生型rpoB可以被逆转录片段取代，从而对抗生素利福平产生耐药性。相反，我们已经证明，rpoB基因的突变可以通过携带该基因的野生型序列的逆转录酶来挽救。

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