Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

Simple item page
dc.contributor.authorLu Linen_US
dc.contributor.authorHouhui Songen_US
dc.contributor.authorYuetong Jien_US
dc.contributor.authorZhili Heen_US
dc.contributor.authorYunting Puen_US
dc.contributor.authorJizhong Zhouen_US
dc.contributor.authorJian Xuen_US
dc.date.accessioned2015-01-23T17:17:30Z
dc.date.accessioned2016-03-30T15:35:56Z
dc.date.available2015-01-23T17:17:30Z
dc.date.available2016-03-30T15:35:56Z
dc.date.issued2010-09-04en_US
dc.descriptionWe thank Yunfeng Yang for helpful discussion and Christoper L. Hemme, Jason Huff and Aifen Zhou for critical reading of the manuscript.en_US
dc.descriptionConceived and designed the experiments: HS. Performed the experiments: LL YJ YP. Analyzed the data: LL HS. Contributed reagents/materials/analysis tools: ZH JZ JX. Wrote the paper: LL HS JX.en_US
dc.description.abstractBackgroundThermophilic, 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 FindingsHere 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 β-1,4-glucanase gene were delivered into X514 with an efficiency of 6×102 transformants/µg 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 β-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.Conclusions/SignificanceIn 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.en_US
dc.description.peerreviewYesen_US
dc.description.peerreviewnoteshttp://www.plosone.org/static/editorial#peeren_US
dc.identifier.citationLin L, Song H, Ji Y, He Z, Pu Y, et al. (2010) Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes. PLoS ONE 5(9): e12582. doi:10.1371/journal.pone.0012582en_US
dc.identifier.doi10.1371/journal.pone.0012582en_US
dc.identifier.urihttp://hdl.handle.net/11244/14064
dc.language.isoen_USen_US
dc.publisherPLos Oneen_US
dc.relation.ispartofseriesPLoS ONE 5(9):e12582en_US
dc.relation.urihttp://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0012582en_US
dc.rightsAttribution 3.0 United Statesen_US
dc.rights.requestablefalseen_US
dc.rights.urihttp://creativecommons.org/licenses/by/3.0/us/en_US
dc.subjectPLOSen_US
dc.subjectPublic Library of Scienceen_US
dc.subjectOpen Accessen_US
dc.subjectOpen-Accessen_US
dc.subjectScienceen_US
dc.subjectMedicineen_US
dc.subjectBiologyen_US
dc.subjectResearchen_US
dc.subjectPeer-reviewen_US
dc.subjectInclusiveen_US
dc.subjectInterdisciplinaryen_US
dc.subjectAnte-disciplinaryen_US
dc.subjectPhysicsen_US
dc.subjectChemistryen_US
dc.subjectEngineeringen_US
dc.titleUltrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobesen_US
dc.typeResearch Articleen_US

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
journal.pone.0012582.pdf
Size:
1.81 MB
Format:
Adobe Portable Document Format
Download

Collections

OU - Faculty and Staff Publications