The Effect of Zinc Oxide and Titanium Dioxide Nanoparticles on Extraction, Purification, Genomic DNA Amplification and Transformation in Some Gram Negative Bacteria

     This study aimed to investigate the effect of using ZnO and TiO nanoparticles on some molecular techniques in order to improve them. The nanoparticles used in this study were characterized using UV/vis spectrophotometer and transmission electron microscope. Results showed that both nanoparticles were highly purified, their shape was spherical and the average diameter was about 100 nm and 50 nm for TiO  and ZnO respectively.  The effect of nanoparticles (TiO  and ZnO) on bacterial growth was determined.  Results illustrated that nanoparticles had antibacterial effect on gram negative bacteria (Escherichia coli HB 101, Pseudomonas aeruginosa). The best antibacterial effect was at concentration of 4mg\ml for TiO and 3 mg/ml for ZnO. The minimum inhibitory concentration (MIC) of ZnO on E. coli HB 101, E. coli MM294 and P. aeruginosa was 0.05, 0.05 and 0.5 mg/ml respectively. While the MIC of TiO on these bacteria was 0.35 mg/ml. Quality and quantity of DNA isolated by boiling, alkali lysis and
salting out methods in presence of nanoparticles were investigated. It was found that ZnO gave a good enhancement of DNA quality and quantity of E. coli and P. aeruginosa. While TiO  led to enhancement of DNA quality for both bacteria by all DNA extraction methods. However, TiO caused enhancement of DNA quantity of both bacteria when used with salting out method only.  Amplifications of lacZ gene of E. coli and 16S rRNA gene of P. aeruginosa were performed in presence of nanoparticles. It was found that ZnO (0.4 mg/ml) was highly enhanced PCR products for both genes.The purity of these products was improved also when using both nanoparticles. Results declared that there is about 50 % reduction on over all PCR reaction time by using both nanoparticles.According to these results, it can be concluded that ZnO might improve the specificity of primers, enhance thermal conductivity of fluid and inhibit restriction enzymes. In order to study the effect of nanoparticles on the transformation, E.coli MM294 was transformed with genomic DNA of E. coli HB 101 and P. aeruginosa. Result indicated that 0.4 mg/ml of ZnO and 0.2 mg/ml of TiO  were enhanced E. coli MM294 transforments. Results reported also that transformation frequency was more increased when MgCl -CaCl  solution replaced by nanoparticles solution, which also reduced the chemical components. Transformation frequencies were increased for about 1.3 and 2.1 folds with TiO  and ZnO respectively,which means that ZnO gave the best results.  To study the effect of nanoparticles on the conjugation. Conjugation experiments were performed between E. coli Hb 101 and P. aeruginosa in presence of nanoparticles. Results showed that there was no effect of any concentration of nanoparticles on conjugation, in which transconjugants still the same with and without using nanoparticles.







2