Volume 18, Issue 3 (7-2024)
payavard 2024, 18(3): 253-263 |
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Ethics code: IR.IAU.KERMAN.REC.1401.076
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Poormehdi M, Khandandezfully N, Amini K. The Molecular Isolation of the srf Gene from Thermophilic Soil Bacilli and Its Cloning in Susceptible Cells for Industry Use. payavard 2024; 18 (3) :253-263
URL: http://payavard.tums.ac.ir/article-1-7556-en.html
URL: http://payavard.tums.ac.ir/article-1-7556-en.html
1- Master of Science in Microbiology, Sirjan Branch, Islamic Azad University, Sirjan, Iran
2- Assistant Professor, Department of Microbiology, Sirjan Branch, Islamic Azad University, Sirjan, Iran ,khandan22@iau.ac.ir
3- Professor, Department of Microbiology, Saveh Branch, Islamic Azad University, Saveh, Iran
2- Assistant Professor, Department of Microbiology, Sirjan Branch, Islamic Azad University, Sirjan, Iran ,
3- Professor, Department of Microbiology, Saveh Branch, Islamic Azad University, Saveh, Iran
Abstract: (89 Views)
Background and Aim: Thermophilic bacillus is a type of thermophilic bacillus, carries various genes and biosurfactants, microbial surfactants are surface active molecules produced by various microorganisms such as bacteria, yeasts and filamentous fungi. Biosurfactants are able to reduce the surface energy between phases and create electrostatic barriers, thus preventing the integration of particles. The aim of the present study was the molecular isolation of the srf gene from thermophilic soil bacilli and its cloning in susceptible cells for industry use.
Materials and Methods: Fifteen samples from different regions of Kerman were collected and screened to isolate Bacillus strains. Morphological and biochemical studies were done to identify the strains. After biochemical examination of isolated microbial isolates and confirmation of Bacillus strains, DNA extraction was done. Then, the srf gene was identified by PCR method from these isolates. The amplified fragment was inserted into pTG19 vector by TA cloning method. Then, the recombinant vector was transformed into E.coli origami bacteria and cloning was confirmed using common methods. The housekeeping gene 16S rRNA was used as the internal control of the test. The analysis of the gene expression level was performed by measuring the relative expression of mRNA as compared to the negative control that E.coli bacteria lacked the srf gene.
Results: A total of 12 isolates of thermophilic bacilli were obtained from soil samples. As result, the PCR reaction for the srf gene with the designed primers was found to be positive in 1 isolate (8.3%). The presence of srf gene and the expression of this gene were checked by real time PCR test. Examining the white and blue colonies, M13 primer, junction location and determination of the 16S rRNA gene sequence confirmed the correctness of the cloning of the mentioned gene in the host bacteria.
Conclusion: As a result of the present study, it was possible to identify the native thermophilic bacillus carrying the srf gene, which can be used to obtain biosurfactant enzyme widely, conveniently and economically, for use in industrial and agricultural purposes, removing oil pollutants and reducing environmental surface tension, etc. which can be advantages.
Materials and Methods: Fifteen samples from different regions of Kerman were collected and screened to isolate Bacillus strains. Morphological and biochemical studies were done to identify the strains. After biochemical examination of isolated microbial isolates and confirmation of Bacillus strains, DNA extraction was done. Then, the srf gene was identified by PCR method from these isolates. The amplified fragment was inserted into pTG19 vector by TA cloning method. Then, the recombinant vector was transformed into E.coli origami bacteria and cloning was confirmed using common methods. The housekeeping gene 16S rRNA was used as the internal control of the test. The analysis of the gene expression level was performed by measuring the relative expression of mRNA as compared to the negative control that E.coli bacteria lacked the srf gene.
Results: A total of 12 isolates of thermophilic bacilli were obtained from soil samples. As result, the PCR reaction for the srf gene with the designed primers was found to be positive in 1 isolate (8.3%). The presence of srf gene and the expression of this gene were checked by real time PCR test. Examining the white and blue colonies, M13 primer, junction location and determination of the 16S rRNA gene sequence confirmed the correctness of the cloning of the mentioned gene in the host bacteria.
Conclusion: As a result of the present study, it was possible to identify the native thermophilic bacillus carrying the srf gene, which can be used to obtain biosurfactant enzyme widely, conveniently and economically, for use in industrial and agricultural purposes, removing oil pollutants and reducing environmental surface tension, etc. which can be advantages.
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