| Summary: | A novel technique for in situ bioremediation is vital to enable the world to meet the need to treat contaminated land; ultrasound gene transfer has that potential. Ultrasound gene transfer has been shown to be a non-invasive, low impact and practical for engineering method of to delivering plasmid DNA and macro-molecules into bacteria. For the first time delivery of a salicylate hydroxylase gene into P. putida UWC1 has been demonstrated, enabling the complete degradation of the salicylate contaminant, which the wild type was unable to degrade, has been demonstrated. Furthermore not only DNA but also macro-molecules (e.g. fluorescent tagged large dextran molecules, up to 2,000,000 MW) have been delivered into P. putida UWC1 using UGT. This can potentially enable delivery of bioparts and nanomaterials for synthetic biology to targeted locations in an organism. To achieve this,: a novel variable frequency ultrasonic generator has been developed to deliver focussed ultrasound through the sonotrode directly into an aqueous bacterial sample. This sonotrode was designed to operate at the optimum frequency for UGT of 27.5 kHz determined using the preliminary apparatus and has enabled the application of UGT to > ~50 ml samples, demonstrating scalability to industrial application (i.e. using an array of sonotrodes to treat litres of environmental sample for re-introduction). The optimum frequency enables a satisfactory rate of transfer (10-7 efficiency) whilst minimising cell lysis (<90% cell survival) making it ideal for environmental application as it will minimise unnecessary disruption to the ecosystem. The mechanism behind UGT has been determined as transfer peaks at the resonant frequencies where cavitation microbubbles are produced. It is the collapse of these microbubbles that generates microjets of extremely high pressure that affect the cell walls of the bacteria enabling uptake of the DNA or macro-molecules. Thus it is shown that the emerging technology of ultrasound gene transfer can deliver novel genes directly into bacteria with minimal preparation and minimal impact to the cells.
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