Efficacy of silver nanoparticles as delivery system in genetic transformation
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Date
2018
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Department of Plant Biotechnology, College of Agriculture, Vellayani
Abstract
The study entitled “Efficacy of silver nanoparticles as a delivery system in genetic transformation” was carried out during 2016-2018 in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to evaluate the efficiency of silver nanoparticles for gene delivery in microbes. In the present study, an assay was conducted in the bacterial strain Escherichia coli DH5α to find out the toxicity of silver nanoparticles. Different concentrations of silver nanoparticles (100 nm size) viz., 0.01, 0.1, 1, 5, 10, 15 and 20 mg L-1 were tried. Maximum inhibition (100%) of growth was observed at the concentrations of 10 mg L-1 and above. 0.01 mg L-1 of silver nanoparticles showed no inhibition of bacterial growth. EC50 of silver nanoparticles was estimated as 4.49 mg L-1 by plotting the graph of inhibition of bacterial cells vs. concentration of silver nanoparticles and carrying out the probability analysis using the statistical package SPSS. Competency was induced in bacterial cells using different concentrations of silver nanoparticles below the EC50 value viz. 0.01, 0.1, 1, 2 and 4 mg L-1 at different time periods (30min, 60min and 120min), silver nanoparticles in combination with 0.1M calcium chloride and 0.1 M calcium chloride alone (conventional method). Bacterial cells without any treatment served as control. Competency induction was assessed by transforming using pUC 18 plasmid vector.
Among the different treatments tried for competency induction, silver nanoparticles showed an increase in transformation efficiency ranging from 2.6 x103 to 8.3x104 cfu/ng of vector DNA compared to the conventional method using 0.1M calcium chloride (2.3 x 103 cfu/ng of vector DNA). Maximum transformation efficiency (8.3x104 cfu/ng of vector DNA) was observed when cells were treated with 1mg L-1 of silver nanoparticles for 60 min.
Treatment with silver nanoparticles for competency induction inhibited the lac Z gene expression of pUC 18 plasmid vector. All the cells treated with silver nanoparticles showed only white colonies on Lauria Bertani (LB) agar plates containing ampicillin (60mg L-1), X-GAL (30mg L-1) and IPTG (80mg L-1). The colonies from cells treated with calcium chloride alone were blue in colour, whereas colonies from cells treated with silver nanoparticles and calcium chloride showed the presence of both white and blue colour. Transformation was reconfirmed by isolation of plasmid from the transformed colonies and checking on agarose gel electrophoresis. The treatment which yielded maximum transformation efficiency (1mg L-1 silver nanoparticles for 60 min) was employed for transforming E. coli cells with two other vectors viz. pBR322 and pCAMBIA. E. coli cells treated with silver nanoparticles (1mg L-1 silver nanoparticles for 60 min) and transformed using pBR322 did not form any colonies on LB agar plates containing ampicillin (60 mg L-1) and tetracycline (20 mg L-1). However, the cells treated with silver nanoparticles grew on LB agar plates containing only ampicillin (60 mg L-1) confirming transformation. Treatment with silver nanoparticles showed an increase in transformation efficiency (8x104 cfu/ng of vector DNA) compared to cells treated with 0.1M calcium chloride (2.3x103cfu/ng of DNA). E. coli cells treated with silver nanoparticles (1mg L-1 for 60 min) and transformed with pCAMBIA vector showed a higher transformation efficiency (7.9 x104 cfu/ng of DNA) compared to calcium chloride method (2.3x103 cfu/ng of DNA) when plated on LB agar plates containing kanamycin (50mg L-1) . Results of the present study indicated that silver nanoparticles aid in bacterial transformation. Silver nanoparticles of 100 nm size at a concentration of 1mg L-1 showed ten fold increase in the transformation efficiency of E. coli DH5α cells with the vectors pUC18, pBR322 and pCAMBIA compared to the conventional method using 0.1 M calcium chloride. Competency induction by silver nanoparticles is economically feasible.
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