| Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes | |
Lin, Lu1; Song, Houhui1; Ji, Yuetong1 ; He, Zhili2; Pu, Yunting1; Zhou, Jizhong2; Xu, Jian1
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| 2010-09-04 | |
| 发表期刊 | PLOS ONE
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| ISSN | 1932-6203 |
| 卷号 | 5期号:9页码:e12582 |
| 摘要 | 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. 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. |
| 文章类型 | Article |
| WOS标题词 | Science & Technology |
| DOI | 10.1371/journal.pone.0012582 |
| 关键词[WOS] | THERMOANAEROBACTER-ETHANOLICUS ; PLASMID DNA ; CLOSTRIDIUM-THERMOSACCHAROLYTICUM ; GENE-TRANSFER ; IN-VIVO ; ELECTROTRANSFORMATION ; STRAIN ; SONOPORATION ; BACTERIA ; ELECTROPORATION |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS研究方向 | Science & Technology - Other Topics |
| WOS类目 | Multidisciplinary Sciences |
| WOS记录号 | WOS:000297726500001 |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qibebt.ac.cn/handle/337004/542 |
| 专题 | 单细胞中心组群 |
| 作者单位 | 1.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 |
| 推荐引用方式 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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