Volume 23, Issue 1 (2020)
mjms 2020, 23(1): 41-48 |
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Jahazi S, Yaghoubi H. Increasing the Efficiency of Gene Transfer in E. coli Using Cationic Carbon Nanotubes. mjms 2020; 23 (1) :41-48
URL: http://mjms.modares.ac.ir/article-30-35505-en.html
URL: http://mjms.modares.ac.ir/article-30-35505-en.html
1- Department of Biology, Ardabil Branch, Islamic Azad University, Ardabil, Iran
2- Department of Biology, Ardabil Branch, Islamic Azad University, Ardabil, Iran , yaghoubi_h@iauardabil.ac.ir
2- Department of Biology, Ardabil Branch, Islamic Azad University, Ardabil, Iran , yaghoubi_h@iauardabil.ac.ir
Abstract: (1837 Views)
Amis: In recent years, carbon nanotubes have attracted the attention of many researchers because of their unique properties. In the present study, carbon nanotubes were coated using PEI. Then, their ability to gene delivery to E. coli cells was examined.
Materials & Methods: Nanotube- PEI nanoparticles were synthesized by the reaction between amine groups of PEI and carboxyl groups of nanotubes. In order to prepare the appropriate DNA vector for delivering to E. coli cells, the Gus A gene was transferred from pBI121 to PUC18 vector (pUC-Gus). Nanotube-PEI/DNA complexes were prepared by combining different mass ratios of nanotube-PEI (0.5, 1, and 2 w/w%) with the fixed amount of DNA. To the transformation of E. coli, the appropriate amount of nanotube-PEI/DNA complexes was added to E. coli cells under stirring at 37°C for 7h. The transformation efficiency of E. coli was determined by colony counting on LB agar supplemented with Ampicillin. Moreover, Gus staining assay was used to confirm the function of the plasmid. Determination of cytotoxicity of nanotube-PEI was performed using MTT assay at 6, 24, and 72 hours intervals at different concentrations of nanotube-PEI (10, 100, and 500μg/ml).
Findings: The nanotube-PEI was synthesized successfully. Nanotube- PEI nanoparticles have a great ability to protect DNA from enzymatic digestion. The percentage of E. coli cells viability was decreased by increasing both the concentration of nanotube-PEI nanoparticles and also the duration of incubation. The results of the agarose gel electrophoresis of plasmid extracted from E. coli and digested using EcoRI enzyme showed that the pUC-Gus plasmid has been successfully transfected by nanotube-PEI nanoparticles to E. coli bacterial cells.
Conclusion: Cationic carbon nanotubes have a high ability to gene transfer to E. coli.
Materials & Methods: Nanotube- PEI nanoparticles were synthesized by the reaction between amine groups of PEI and carboxyl groups of nanotubes. In order to prepare the appropriate DNA vector for delivering to E. coli cells, the Gus A gene was transferred from pBI121 to PUC18 vector (pUC-Gus). Nanotube-PEI/DNA complexes were prepared by combining different mass ratios of nanotube-PEI (0.5, 1, and 2 w/w%) with the fixed amount of DNA. To the transformation of E. coli, the appropriate amount of nanotube-PEI/DNA complexes was added to E. coli cells under stirring at 37°C for 7h. The transformation efficiency of E. coli was determined by colony counting on LB agar supplemented with Ampicillin. Moreover, Gus staining assay was used to confirm the function of the plasmid. Determination of cytotoxicity of nanotube-PEI was performed using MTT assay at 6, 24, and 72 hours intervals at different concentrations of nanotube-PEI (10, 100, and 500μg/ml).
Findings: The nanotube-PEI was synthesized successfully. Nanotube- PEI nanoparticles have a great ability to protect DNA from enzymatic digestion. The percentage of E. coli cells viability was decreased by increasing both the concentration of nanotube-PEI nanoparticles and also the duration of incubation. The results of the agarose gel electrophoresis of plasmid extracted from E. coli and digested using EcoRI enzyme showed that the pUC-Gus plasmid has been successfully transfected by nanotube-PEI nanoparticles to E. coli bacterial cells.
Conclusion: Cationic carbon nanotubes have a high ability to gene transfer to E. coli.
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