Engineering E. coli strains using antibiotic-resistance-gene-free plasmids.
Engineering E. coli strains using antibiotic-resistance-gene-free plasmids.
复制标题
利用无抗生素抗性基因的质粒构建大肠杆菌菌株。
DOI：
10.1016/j.crmeth.2023.100669
复制
发表时间：
2023-12-18
期刊：
影响因子：
--
通讯作者：
Moon, Tae Seok
中科院分区：
文献类型：
--
作者：
Amrofell, Matthew B.;Rengarajan, Sunaina;Vo, Steven T.;Tovar, Erick S. Ramirez;Lobello, Larissa;Dantas, Gautam;Moon, Tae Seok
我们使用营养缺陷型和必需基因双重选择策略，为基于抗生素耐药无基因(ARGFP)的克隆创建了一条可推广的管道。我们使用营养缺陷型选择在工程菌DH10B克隆菌株中构建质粒，并使用基于营养缺陷型和必需基因的选择来(1)选择重组菌株，(2)在工程益生菌应用的通用底盘E.ColiNissle 1917和实验室的野生型E.ColiMG1655中保持质粒。我们证明了我们的方法与基于抗生素抗性基因的克隆方法具有相当的效率。我们还表明，双敲除Nissle和MG1655菌株很容易用感兴趣的质粒转化。值得注意的是，我们表明，工程尼斯勒菌株在重复培养中以及在小鼠肠道中都能长期保持质粒，展示了广泛应用的潜力，同时将通过水平基因转移传播抗生素耐药性的风险降至最低。我们建立了一种基于ARGFP的克隆方法，ARGFP在体外可以在没有选择压力的情况下保持一个月，ARGFP在小鼠肠道中表现出长期的维持。我们将ARGFP方法应用于多个菌株，表明其通用性好的质粒是基因工程必不可少的工具。然而，目前使用抗生素耐药性基因来维持细胞群体中的质粒有传播抗生素耐药性的风险，特别是在不受控制的环境中。在这里，我们提出了可用于克隆并保存在正在进行长期传代或已在小鼠肠道定植的工程菌中的抗生素耐药无基因质粒(ARGFP)。抗生素耐药基因(ARGs)的广泛使用和不完全处置会通过水平基因转移增加抗生素耐药性的传播。阿姆罗费尔等人。开发一种不含ARG的、基于质粒的克隆方法，证明这种质粒在体外和体内的长期保持，并使用大肠杆菌展示其普适性。
We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection strategy. We use auxotrophic selection to construct plasmids in engineered E. coli DH10B cloning strains and both auxotrophic- and essential-gene-based selection to (1) select for recombinant strains and (2) maintain a plasmid in E. coli Nissle 1917, a common chassis for engineered probiotic applications, and E. coli MG1655, the laboratory “wild-type” E. coli strain. We show that our approach has comparable efficiency to that of antibiotic-resistance-gene-based cloning. We also show that the double-knockout Nissle and MG1655 strains are simple to transform with plasmids of interest. Notably, we show that the engineered Nissle strains are amenable to long-term plasmid maintenance in repeated culturing as well as in the mouse gut, demonstrating the potential for broad applications while minimizing the risk of antibiotic resistance spread via horizontal gene transfer. We develop an antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning method ARGFPs are maintained without selection pressure for a month in vitro ARGFPs show long-term maintenance in the mouse gut We apply the ARGFP method to multiple strains, showing its generalizability Plasmids are essential tools of genetic engineering. However, the current use of antibiotic resistance genes to maintain plasmids in cellular populations risks the spread of antibiotic resistance, particularly in uncontrolled environments. Here, we present antibiotic-resistance-gene-free plasmids (ARGFPs) that can be used for cloning and maintained in engineered strains of E. coli that are undergoing extended subculturing or that have colonized the murine gut. Widespread use and incomplete disposal of antibiotic resistance genes (ARGs) can increase antibiotic resistance spread via horizontal gene transfer. Amrofell et al. develop an ARG-free, plasmid-based cloning method, demonstrate the long-term maintenance of such plasmids in vitro and in vivo, and show its generalizability using E. coli.
登录
查看更多内容
影响因子：
4.7
作者：
Kan A;Gelfat I;Emani S;Praveschotinunt P;Joshi NS
通讯作者：
Joshi NS
影响因子：
8.4
作者：
Kang, Chae Won;Lim, Hyun Gyu;Jung, Gyoo Yeol
通讯作者：
Jung, Gyoo Yeol
影响因子：
9.3
作者：
Noto Guillen M;Rosener B;Sayin S;Mitchell A
通讯作者：
Mitchell A
影响因子：
4.1
作者：
Hägg, P;de Pohl, JW;Isaksson, LA
通讯作者：
Isaksson, LA
影响因子：
16.6
作者：
Hwang IY;Koh E;Wong A;March JC;Bentley WE;Lee YS;Chang MW
通讯作者：
Chang MW