Low Frequency And Low Intensity Ultrasound Mediated Plasmid Transfer Into Escherichia Coli And Mycobacterium Smegmatis

It is a new method for ultrasound promoting the transformation or transfection of gene.Ultrasound-mediated transformation almost relied on medical biology,which means the smaller biological damage,the simpler method of operation.Ultrasound conversion can be completed with the same and larger experimental conditions,so as to developing the most umique advantage in industrial producyion.Escherichia coli DH5? and Mycobacterium smegmatis were used as recipient bacteria.Escherichia coli DH5?,Gram-negative bacterium,was used in gene transformation.Mycobacterium smegmatis,Cell wall structure is not only special,difficult to transform,but also is an acid-resistant positive bacterium.It is established a method in which low-frequency and low-intensity ultrasound?LFLIU?mediates to the transfer of plasmid DNA into bacteria conveniently,stably and efficiently.In the future,ultrasound-mediated transformation may be a promising adjunct technique for gene therapy.ObjectiveTo investigate the influence of the variety of factors?such as ra diation intensity,irradiation time,CaCl₂ concentration,microbubble co ncentration?oflow frequency and low intensity ultrasound?LFLIU?mediatedplasmidtransferringinto bacteria.The plasmid expressed the ge ne and protein level.Methods1.A mixture of E.coli and plasmid were exposed to 42 kHz ultrasound.The experimental conditions were followed by:?1?0.13 W/cm² ultrasound continuously irradiate 0 s,20 s,40 s,80 s,160 s,320 s with 0.5 mmol/L CaCl₂ solution for ultrasonic medium,?2?0.51 W/cm² ultrasound irradiate 160 s,with sterile saline?not containing Ca2+?,0.5 mmol/L CaCl₂,1 mmol/L CaCl₂ mmol/L,5 CaCl₂,10 mmol/L CaCl₂ solution for the ultrasonic medium;?3?0 W/cm²,0.13 W/cm²,0.35 W/cm²,0.51 W/cm²,0.72 W/cm² ultrasound irradiate 160 s,with 1 mmol/L CaCl₂.Observation:?1?The different conditions of transfer efficiency were grouping;?2?The survival rate of E.coli was detected with the intensity of ultrasonic aforementioned by flow cytometry;?3?Plasmid integrity was tested by agarose gel electrophoresis.2.The effect of LFLIU combined with microbubbles promote plasmid was transferred into Mycobacterium smegmatis: 40 kHz,0.27W/cm² ultrasonic bath irradiate Mycobacterium smegmatis and exogenous substances mixed liquid with Sonovue microbubble concentration on 0 mg/ml,1.25 mg/ml,2.5 mg/ml,3.75 mg/ml in 0 min,3 min,5 min,10 min,20 min,separately.Observation:?1?the different conditions of transfer efficiency under different conditions was detected;?2?the influence of the survival rate of Mycobacterium smegmatis was detected by colony counting method;?3?Dextran-FITC fluorescence was detected by confocal microscopy after LFLIU irradiation into bacteria;?4?Scanning electron microscopy was used to observe the microstructure of Mycobacterium after ultrasonic irradiation.?5?The expression ClpP2 gene was analyzed by q-PCR and Western blot detection.Results1.Positive clonies were selected on the agarose plate containing antibiotics after ultrasound exposing.The efficiency of plasmid transformation was related to the dose of ultrasonic irradiation,the concentration of CaCl₂ and the concentration of microbubbles.2.LFLIU combined with the Sonovue? cansignificantly.improve the efficiency of the transformation plasmid.The transfer efficiency is 19.33× 10²CFU/?g DNA,which means more than19 times in effect of ultrasound had been developed.3.The electrophoresis detection showed that the configurations of plasmid were changed through the higher intensity of ultrasound,but the integrity was not yet changed.4.Scanning electron microscopy showed that the damage of bacteria was smaller after ultrasonic irradiation alone.The ultrasound combined with microbubbles had some damage to ultrasound,resulting in swelling of bacteria,breakage of cell walls,liquefaction and death of bacteria.5.There were no significant differences between the level of gene and protein expression by q-PCR and Western blot.ConclusionsLFLIU can mediate plasmid transrring into bacteria efficiently and safely.Moreover,ultrasound combined with microbubbles can dramatically increase the efficiency of plasmid transformation.Although the intensity of ultrasound had not yet altered,the configurations of plasmid were dramatically transformed.However,there were no significant differences between the level of gene and protein expression.