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Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes
Lin, Lu1; Song, Houhui1; Ji, Yuetong1; He, Zhili2; Pu, Yunting1; Zhou, Jizhong2; Xu, Jian1
2010-09-04
Source PublicationPLOS ONE
ISSN1932-6203
Volume5Issue:9Pages:e12582
AbstractThermophilic, 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. 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 beta-1,4-glucanase gene were delivered into X514 with an efficiency of 6x10(2) transformants/g of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by visualization of fluorescence signals of secondary host-cells following a plasmid-rescue experiment. Furthermore, the foreign beta-1,4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic. In this study, we developed an ultrasound-based sonoporation method in TGPAs. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically.; 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.
SubtypeArticle
WOS HeadingsScience & Technology
DOI10.1371/journal.pone.0012582
WOS KeywordTHERMOANAEROBACTER-ETHANOLICUS ; PLASMID DNA ; CLOSTRIDIUM-THERMOSACCHAROLYTICUM ; GENE-TRANSFER ; IN-VIVO ; ELECTROTRANSFORMATION ; STRAIN ; SONOPORATION ; BACTERIA ; ELECTROPORATION
Indexed BySCI
Language英语
WOS Research AreaScience & Technology - Other Topics
WOS SubjectMultidisciplinary Sciences
WOS IDWOS:000297726500001
Citation statistics
Document Type期刊论文
Identifierhttp://ir.qibebt.ac.cn/handle/337004/542
Collection单细胞中心组群
Affiliation1.Chinese Acad Sci, Qingdao Inst BioEnergy & Bioproc Technol, Qingdao, Shandong, Peoples R China
2.Univ Oklahoma, Dept Bot & Microbiol, Inst Environm Genom, Norman, OK 73019 USA
Recommended Citation
GB/T 7714
Lin, Lu,Song, Houhui,Ji, Yuetong,et al. Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes[J]. PLOS ONE,2010,5(9):e12582.
APA Lin, Lu.,Song, Houhui.,Ji, Yuetong.,He, Zhili.,Pu, Yunting.,...&Xu, Jian.(2010).Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes.PLOS ONE,5(9),e12582.
MLA Lin, Lu,et al."Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes".PLOS ONE 5.9(2010):e12582.
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