Development of constitutive and IPTG-inducible integron promoter-based expression systems for Escherichia coli and Agrobacterium tumefaciens

• Published in: 3 Biotech 2023-03, Vol.13 (3), p.91-91, Article 91
• Main Authors: Toh, Wai Keat, Teo, Yuh Leng, Tor, Xin Yen, Loh, Pek Chin, Wong, Hann Ling
• Format: Article
• Language: English
• Subjects: Agriculture; Agrobacterium; Agrobacterium tumefaciens; Antibiotic resistance; Bacteria; Bioinformatics; Biomaterials; Biotechnology; Cancer Research; Chemistry; Chemistry and Materials Science; E coli; Escherichia coli; Expression vectors; Fluorescence; Gene expression; Genetic transformation; Gram-negative bacteria; Green fluorescent protein; Host range; Molecular biology; Original; Original Article; Promoters; Proteins; Reporter gene; Stem Cells; Transgenes
• ISSN: 2190-572X; 2190-5738
• DOI: 10.1007/s13205-023-03507-0

Broad host range (BHR) expression vector is a vital tool in molecular biology research and application. Currently, most of the plasmid vectors used in Agrobacterium spp. are binary vectors that are designed for plant transformation, and very few are designed for expressing transgenes in Agrobacterium 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, PcS  +  P2 , 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” ( P int ) henceforth. Herein, we created two versions of isopropyl– d -thiogalactopyranoside (IPTG)-inducible promoters by substituting and/or inserting lac O sequence(s) into P int . 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 Escherichia coli and Agrobacterium tumefaciens . Functional validation of these vectors was performed by the expression of the reporter gene, superfolder green fluorescent protein ( sfGFP ), 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 E. coli and A. tumefaciens , thus facilitating recombinant protein production and genetic studies in Gram-negative bacteria.