Toh Wai Keat, Teo Yuh Leng, Tor Xin Yen, Loh Pek Chin, Wong Hann Ling
Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak Malaysia.
3 Biotech. 2023 Mar;13(3):91. doi: 10.1007/s13205-023-03507-0. Epub 2023 Feb 20.
Broad host range (BHR) expression vector is a vital tool in molecular biology research and application. Currently, most of the plasmid vectors used in spp. are binary vectors that are designed for plant transformation, and very few are designed for expressing transgenes in spp. Class 1 integrons are common genetic elements that allow for the efficient capture and expression of antibiotic resistance genes, especially in Gram-negative bacteria. One of its compound promoters, + , was used in this study and has been reported to be the strongest class 1 integron constitutive promoter; it is referred to as "integron promoter" ( ) henceforth. Herein, we created two versions of isopropyl-d-thiogalactopyranoside (IPTG)-inducible promoters by substituting and/or inserting O sequence(s) into . These inducible promoters, which possess different degrees of stringency and inducibility, were used to construct two broad host range expression vectors (pWK102 and pWK103) based on the versatile pGREEN system. This allows them to be stably maintained and replicated in both and . Functional validation of these vectors was performed by the expression of the reporter gene, superfolder green fluorescent protein (), which was cloned downstream of these promoters. Due to the strong induction and tunable expression of a transgene located downstream to the inducible integron promoter, these vectors may be useful for heterologous gene expression in both and , thus facilitating recombinant protein production and genetic studies in Gram-negative bacteria.
The online version contains supplementary material available at 10.1007/s13205-023-03507-0.
广泛宿主范围（BHR）表达载体是分子生物学研究和应用中的重要工具。目前，用于[具体物种1]的大多数质粒载体是为植物转化设计的二元载体，而专门为在[具体物种2]中表达转基因设计的很少。1类整合子是常见的遗传元件，可高效捕获和表达抗生素抗性基因，尤其是在革兰氏阴性菌中。本研究使用了其复合启动子之一，即[具体启动子名称1]+[具体启动子名称2]，据报道它是最强的1类整合子组成型启动子；此后将其称为“整合子启动子”（[具体缩写]）。在此，我们通过将O序列替换和/或插入[具体启动子名称1]中，创建了两个异丙基 - d - 硫代半乳糖苷（IPTG）诱导型启动子版本。这些具有不同严格程度和诱导性的诱导型启动子，用于基于通用pGREEN系统构建两个广泛宿主范围表达载体（pWK102和pWK103）。这使得它们能够在[具体物种1]和[具体物种2]中稳定维持和复制。通过在这些启动子下游克隆报告基因超折叠绿色荧光蛋白（[具体蛋白名称]），对这些载体进行了功能验证。由于位于诱导型整合子启动子下游的转基因具有强诱导性和可调节表达，这些载体可能有助于在[具体物种1]和[具体物种2]中进行异源基因表达，从而促进革兰氏阴性菌中的重组蛋白生产和遗传研究。
在线版本包含可在10.1007/s13205 - 023 - 03507 - 0获取的补充材料。
