Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene.

Lixia FU; Jingxiao XU; Xian'gan Han; Hui Yang; Yingtiao Lai; Zhibin Huang; Jiansen Gong

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Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene.

Author: Lixia FU ¹ ; Jingxiao XU ² ; Xian'gan HAN ³ ; Hui YANG ⁴ ; Yingtiao LAI ¹ ; Zhibin HUANG ¹ ; Jiansen GONG ²
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1. Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, Guangdong, China.
2. Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou 225125, Jiangsu, China.
3. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
4. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu, China.
Publication Type:Journal Article
Keywords: arabinose promoter; pUC replicon; promoter reporter system; reporter gene; site-directed mutagenesis; β-galactosidase
MeSH: Cloning, Molecular; Escherichia coli/genetics*; Genes, Reporter/genetics*; Genetic Vectors/genetics*; Lac Operon/genetics*; Plasmids/genetics*; beta-Galactosidase/genetics*
From: Chinese Journal of Biotechnology 2021;37(1):321-330
CountryChina
Language:Chinese
Abstract: To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.