We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.

中文翻译：

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大肠杆菌中新陈代谢和核苷三磷酸摄取的定向进化

我们报告设计和精心设计的选择协议，用于在大肠杆菌中导入DNA聚合酶，三磷酸胸苷（dTTP）的典型底物。通过从插入到染色体中的合成基因表达的藻类质体转运蛋白的作用，构建了生长依赖于dTTP摄取的细菌菌株，并显示出可同时承受胸苷酸合酶和胸苷激酶的损失。这样的tdk双重缺失菌株为防止微生物转基因生物的传播提供了严格的营养控制的实验模型。我们的菌株按照以下优先顺序运输了四个典型的dNTP：dCTP> dATP≥dGTP> dTTP。在外源性dTTP限制下的长期培养导致dNTP转运通过适应性进化而增强。我们研究了糖或核碱基部分改变对dCTP类似物的摄取，发现它们分别引起细胞活力丧失和突变频率增加。配备有核苷三磷酸转运蛋白的大肠杆菌菌株应有助于进化的生物，其DNA基因组可以通过完全取代其规范的核苷酸成分而化学修饰。