Development of expression vector with the Pgrac100 promoter induced by IPTG and integrated gfp+ gene into Bacillus subtilisgenome at lacA locus

Ministry of Science and Technology, Vietnam Pub Date : 2024-01-25 DOI:10.31276/vjst.66(1).75-80

Thi Bich Phuong Chu, D. Nguyen, Thi Phuong Trang Phan

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Abstract

Bacillus subtilis is a prospective gram - positive bacterium that offers an opportunity for the production of recombinant proteins in the pharmaceutical, agricultural, industrial, and food industries.The goal of this study was to develop an integrative inducible expression system by incorporating the Pgrac100 promoter into the lacA locus of the B. subtilis genome. The results showed that the Pgrac100 promoter was able to strictly control the expression of the target protein, with induction factors of 3.3, 7.6, and 10.8 after 4 hours under induction with 0.01, 0.1, and 1 mM Isopropulβ-D-1-thiogalactopyranoside (IPTG), respectively. The duration of induction and the concentration of IPTG had significant effects on the efficacy of target protein expression. Specifically, when induced with 0.1 mM IPTG after 4 hours, there was a significant rise in GFP expression, accounting for 13% of the total cellular protein. Our new vector system provided more options for the production of recombinant proteins in basic research and industrial applications.

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开发带有 IPTG 诱导的 Pgrac100 启动子的表达载体，并将 gfp+ 基因整合到枯草芽孢杆菌基因组的 lacA 基因座上

本研究的目的是通过将Pgrac100启动子整合到枯草杆菌基因组的lacA基因座中，开发一种整合诱导表达系统。结果表明，Pgrac100启动子能够严格控制目标蛋白的表达，在0.01、0.1和1 mM异丙β-D-1-硫代半乳糖苷（IPTG）诱导下，4小时后的诱导因子分别为3.3、7.6和10.8。诱导时间的长短和 IPTG 的浓度对目标蛋白的表达效果有显著影响。具体来说，用 0.1 mM IPTG 诱导 4 小时后，GFP 的表达量显著上升，占细胞蛋白总量的 13%。我们的新载体系统为基础研究和工业应用中重组蛋白的生产提供了更多选择。

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Ministry of Science and Technology, Vietnam

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