Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis.
Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis.
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用于诱导双蛋白表达的单质粒系统和乳酸菌乳酸菌中的CRISPR-CAS9/CRISPR-CAS9/CRISPRI基因调节。
DOI：
10.1038/s41598-018-19402-1
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发表时间：
2018-01-17
影响因子：
4.6
通讯作者：
Štrukelj B
中科院分区：
文献类型：
--
作者：
Berlec A;Škrlec K;Kocjan J;Olenic M;Štrukelj B
乳酸乳球菌是一种食品级乳酸菌，在乳品工业中用作细胞工厂和重组蛋白表达的宿主。乳链菌肽控制的诱导表达系统（NICE）是乳杆菌中应用最广泛的表达系统。lactis;然而，用于其遗传修饰的新工具是高度期望的。在这项工作中，NICE适用于双蛋白表达。构建了含有两个乳链菌肽启动子和多克隆位点（MCSs）的质粒pNZDual和含有单个乳链菌肽启动子和两个由核糖体结合位点分开的MCSs的质粒pNZPolycist。以所有可能的组合克隆红外荧光蛋白和人IgG结合DARPin的基因以评估蛋白质产量。双启动子质粒pNZDual能够平衡表达两种模型蛋白。它被开发用于通过使用乳链菌肽启动子开发单质粒诱导型CRISPR-Cas9系统（pNZCRISPR），首先驱动Cas9表达，其次驱动单向导RNA转录。针对htrA和ermR的sgRNA引导Cas9针对基因组或质粒DNA，并引起细菌生长和存活的变化。用dCas 9替换Cas9使得CRISPR干扰介导的upp基因沉默成为可能。本研究介绍了一系列用于乳酸菌遗传改良的质粒。乳。
Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.
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影响因子：
3.8
作者：
Luo ML;Leenay RT;Beisel CL
通讯作者：
Beisel CL
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
2.6
作者：
Pérez-Arellano, I;Zúñiga, M;Pérez-Martínez, G
通讯作者：
Pérez-Martínez, G
影响因子：
2.2
作者：
Herigstad, B;Hamilton, M;Heersink, J
通讯作者：
Heersink, J
DOI：
10.1099/13500872-141-8-1883
发表时间：
1995-08-01
期刊：
MICROBIOLOGY-UK
影响因子：
--
作者：
MARTINUSSEN, J;HAMMER, K
通讯作者：
HAMMER, K