Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions.

中文翻译：

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SynMyco转座子：工程转座子载体，可有效转化最少的基因组。

支原体是系统和合成生物学的重要模式生物，对多种物种都有致病性。尽管它们具有相关性，但许多其他微生物中用于基因组编辑的工具却无法用于支原体。Tn4001转座子是处理这些细菌的参考工具，但是报道的不同物种的转化效率（TEs）差异很大。在这里，我们探讨了四种支原体物种（无乳支原体，铁支原体支原体，鸡支原体和肺炎支原体）中这些差异的潜在机制，这些支原体被选为支原体属每个簇的代表成员。我们发现驱动转座酶和抗生素抗性标志物表达的调控区（RR）对TEs有重大影响。然后，我们设计了称为SynMyco RR的合成RR，以控制关键转座子载体元件的表达。使用这种合成的RR，我们能够将鸡毒支原体，feriruminatoris和无乳支原体的TE分别提高30倍，980倍和1036倍。最后，为了说明这种新转座子的潜力，我们在无乳分枝杆菌中进行了第一个必需性研究，该研究基于199,000多个基因组插入。