Cloning of Bacillus subtilis phytase gene construct in Escherichia coli

Mahdiyar Iravani Saadi; Abbas Doosti; Heeva Jalali; Ehsan Nabi Abdolyousefi; Mansooreh Hooshiyar; Reza Tabrizi; Esmat Noshadi

doi:10.18502/ijm.v13i5.7433

Mahdiyar Iravani Saadi Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Abbas Doosti Biotechnology Research Center, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
Heeva Jalali Department of Animal Science, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran
Ehsan Nabi Abdolyousefi Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Mansooreh Hooshiyar Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
Reza Tabrizi Non-Communicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran
Esmat Noshadi Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

DOI: https://doi.org/10.18502/ijm.v13i5.7433

Keywords: Bacillus subtilis; Cloning; Escherichia coli; Phytase; Probiotics

Abstract

Background and Objectives: Phytase has a hydrolysis function of phytic acid, which yields inorganic phosphate. Bacillus species can produce thermostable alkaline phytase. The aim of this study was to isolate and clone a Phytase gene (Phy) from Bacillus subtilis in Escherichia coli.

Materials and Methods: In this study, the extracellular PhyC gene was isolated from Bacillus subtilis Phytase C. After purification of the bands, DNA fragment of Phy gene was cloned by T/A cloning technique, and the clone was transformed into Escherichia coli. Afterward, the pGEM-Phy was transferred into E. coli Top-10 strain and the recombinants were plated on LB agar containing 100 µg/ml ampicillin. The colonization of 1171 bp of gene Phytase C was confirmed by PCR. The presence of gene-targeting in vector was confirmed with enzymatic digestion by XhoI and XbaI restriction enzymes.

Results: The Phytase gene was successfully cloned in E. coli. The result of cloning of 1171 bp Phytase gene was confirmed by PCR assay.

Conclusion: Our impression of this article is that several methods, such as using along with microbial, plant phytase reproduction, or low-phytic acid corn may be the better way from a single phytase.