Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid, 28049, Spain.
ACS Synth Biol. 2021 Apr 16;10(4):690-697. doi: 10.1021/acssynbio.0c00611. Epub 2021 Mar 22.
Chromosomal exchange and subsequent recombination of the cognate DNA between bacteria was one of the most useful genetic tools (e.g., Hfr strains) for genetic analyses of before the genomic era. In this paper, yeast assembly has been used to recruit the conjugation machinery of environmentally promiscuous RP4 plasmid into a minimized, synthetic construct that enables transfer of chromosomal segments between donor/recipient strains of KT2440 and potentially many other Gram-negative bacteria. The synthetic device features [i] a R6K suicidal plasmid backbone, [ii] a mini-Tn5 transposon vector, and [iii] the minimal set of genes necessary for active conjugation (RP4 Tra1 and Tra2 clusters) loaded as cargo in the mini-Tn5 mobile element. Upon insertion of the transposon in different genomic locations, the ability of -TRANS (ransference of P4-ctivated ucleotide egments) donor strains to mobilize genomic stretches of DNA into neighboring bacteria was tested. To this end, a double mutant Δ (uracil auxotroph) Δ (unable to grow on glucose) was used as recipient in mating experiments, and the restoration of the / phenotypes allowed for estimation of chromosomal transfer efficiency. Cells with the inserted transposon behaved in a manner similar to Hfr-like strains and were able to transfer up to 23% of their genome at frequencies close to 10 exconjugants per recipient cell. The hereby described TRANS device not only expands the molecular toolbox for , but it also enables a suite of genomic manipulations which were thus far only possible with domesticated laboratory strains and species.
在基因组时代之前，细菌之间同源 DNA 的染色体交换和随后的重组是最有用的遗传工具之一（例如，Hfr 菌株），可用于遗传分析。在本文中，酵母组装已被用于招募环境杂合性 RP4 质粒的接合机制进入一个最小化的合成构建体，该构建体能够在供体/受体菌株之间转移染色体片段 KT2440 和可能许多其他革兰氏阴性细菌。该合成装置的特点是：[i]一个 R6K 自杀质粒骨架，[ii]一个 mini-Tn5 转座子载体，和 [iii]一个最小的一组基因，这些基因对于活性接合（RP4 Tra1 和 Tra2 簇）是必需的，作为 mini-Tn5 移动元件中的货物加载。在不同基因组位置插入转座子时，测试了 -TRANS（转移 P4 激活核苷酸片段）供体菌株将基因组 DNA 大片段动员到邻近细菌中的能力。为此，使用 double mutant Δ（尿嘧啶营养缺陷型）Δ（不能在葡萄糖上生长）作为交配实验中的受体，/ 表型的恢复允许估计染色体转移效率。插入转座子的细胞表现出类似于 Hfr 样菌株的行为，并且能够以接近每受体细胞 10 个外共轭子的频率转移高达 23%的基因组。本文描述的 TRANS 装置不仅扩展了 的分子工具包，而且还能够进行一系列基因组操作，迄今为止，这些操作仅在驯化的实验室菌株和物种中才有可能。
