Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation.
Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation.
复制标题
ColE1 质粒稳定性的遗传控制独立于质粒拷贝数调节。
DOI：
10.1007/s00294-018-0858-0
复制
发表时间：
2019-03
期刊：
影响因子：
2.5
通讯作者：
Camps M
中科院分区：
文献类型：
--
作者：
Standley MS;Million-Weaver S;Alexander DL;Hu S;Camps M
类Cole1载体广泛用于重组基因在大肠杆菌中的表达。对于这些载体来说，在细胞分裂过程中，单个质粒分离到子代细胞似乎是随机的，当没有确保它们维持的机制时，随着时间的推移，它们很容易丢失。在这里，我们使用在recA relA菌株中的pGFPuv作为致敏模型，在重组基因表达的背景下研究影响质粒稳定性的因素。我们发现，在这个模型中，可以通过对复制起点(ORI)序列进行两种类型的遗传修饰来恢复质粒的稳定性：点突变和在质粒ORI的5‘端新的269个核苷酸重复，我们称之为DAS(复制反义)ORI。这些修饰的组合产生了一定范围的拷贝数和重组表达水平。与经典的随机分布模型直接矛盾的是，我们发现增加的质粒拷贝数与增加的质粒稳定性之间没有相关性。稳定性的提高也不能用重组基因表达水平的降低来解释。我们的观察结果将更符合混合聚集和自由分布模型，该模型是最近提出的，该模型是基于使用超分辨率荧光显微镜在体内检测单个质粒而提出的。这项工作表明，质粒ORI在控制ColE1质粒分离方面发挥了不同于复制启动的作用，为旨在增强大规模重组基因表达或生物修复的策略中对质粒稳定性的遗传调控打开了大门。
ColE1-like plasmid vectors are widely used for expression of recombinant genes in E. coli. For these vectors, segregation of individual plasmids into daughter cells during cell division appears to be random, making them susceptible to loss over time when no mechanisms ensuring their maintenance are present. Here we use the plasmid pGFPuv in a recA relA strain as a sensitized model to study factors affecting plasmid stability in the context of recombinant gene expression. We find that in this model, plasmid stability can be restored by two types of genetic modifications to the plasmid origin of replication (ori) sequence: point mutations and a novel 269 nt duplication at the 5’ end of the plasmid ori, which we named DAS (Duplicated Anti-Sense) ori. Combinations of these modifications produce a range of copy numbers and of levels of recombinant expression. In direct contradiction with the classic random distribution model, we find no correlation between increased plasmid copy number and increased plasmid stability. Increased stability cannot be explained by reduced levels of recombinant gene expression either. Our observations would be more compatible with a hybrid clustered and free distribution model, which has been recently proposed based on detection of individual plasmids in vivo using super-resolution fluorescence microscopy. This work suggests a role for the plasmid ori in the control of segregation of ColE1 plasmids that is distinct from replication initiation, opening the door for the genetic regulation of plasmid stability as a strategy aimed at enhancing large-scale recombinant gene expression or bioremediation.
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DOI：
10.1073/pnas.77.5.2450
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
ITOH, T;TOMIZAWA, J
通讯作者：
TOMIZAWA, J
影响因子：
3.3
作者：
Lili, Loukia N.;Britton, Nicholas F.;Feil, Edward J.
通讯作者：
Feil, Edward J.
影响因子：
2.7
作者：
Kitsera, Natahya;Khobta, Andriy;Epe, Bernd
通讯作者：
Epe, Bernd
影响因子：
4.9
作者：
Li, Jun-Jie;Spychala, Caressa N.;Doi, Yohei
通讯作者：
Doi, Yohei
DOI：
10.1007/bf00273210
发表时间：
1979-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
OHMORI, H;TOMIZAWA, JI
通讯作者：
TOMIZAWA, JI