《Ultrasound-Mediated DNA__ Transformation in》.pdfVIP

Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes 1. 1. 1 2 1 2 1 Lu Lin , Houhui Song , Yuetong Ji , Zhili He , Yunting Pu , Jizhong Zhou *, Jian Xu * 1 Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China, 2 Institute For Environmental Genomics, Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma, United States of America Abstract Background: Thermophilic, Gram-positive, anaerobic bacteria (TGPAs) are generally recalcitrant to chemical and electrotransformation due to their special cell-wall structure and the low intrinsic permeability of plasma membranes. Methodology/Principal Findings: Here we established for any Gram-positive or thermophiles an ultrasound-based sonoporation as a simple, rapid, and minimally invasive method to genetically transform TGPAs. We showed that by applying a 40 kHz ultrasound frequency over a 20-second exposure, Texas red-conjugated dextran was delivered with 27% efficiency into Thermoanaerobacter sp. X514, a TGPA that can utilize both pentose and hexose for ethanol production. Experiments that delivered plasmids showed that host-cell viability and plasmid DNA integrity were not compromised. Via sonoporation, shuttle vectors pHL015 harboring a jellyfish gfp gene and pIKM2 encoding a Clostridium thermocellum b-1,4- glucanase gene were delivered into X514 with an efficiency of 6 6102 transformants/mg of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by