Plasmid最新文献_第10页

Cloning of pAhX22, a small cryptic plasmid from Aeromonas hydrophila, and construction of a pAhX22-derived shuttle vector 嗜水气单胞菌小隐质粒pAhX22的克隆及pAhX22穿梭载体的构建

IF 2.6 4区生物学

Plasmid Pub Date : 2020-03-01 DOI: 10.1016/j.plasmid.2020.102490

Xingyu Kang , Chunqiu Li , Yi Luo

{"title":"Cloning of pAhX22, a small cryptic plasmid from Aeromonas hydrophila, and construction of a pAhX22-derived shuttle vector","authors":"Xingyu Kang , Chunqiu Li , Yi Luo","doi":"10.1016/j.plasmid.2020.102490","DOIUrl":"10.1016/j.plasmid.2020.102490","url":null,"abstract":"<div>In this study, a cryptic plasmid from Aeromonas hydrophila (pAhX22) was cloned and characterized. pAhX22 was 2523 bp long, had a GC content of 59.9%, and contained two putative open reading frames (ORFs). orf1 and orf2 encoded putative proteins of 458 amino acids and 88 amino acids, respectively; these putative proteins might be involved in plasmid replication. An Escherichia coli–A. hydrophila shuttle vector, pAEsv-1 (4587 bp, KanR), was constructed using in-fusion cloning, combining pAhX22 with the kanamycin-resistance gene and the origin of replication from E. coli expression vector pET-28a. The transformation efficiency of pAEsv-1 in A. hydrophila strains ranged from 2.2 × 106 to 1.0 × 107 CFU/μg DNA, while transformation efficiency in E. coli DH5α was about 1.6 × 106 CFU/μg DNA. pAEsv-1 was segregationally and structurally stable in A. hydrophila in the absence of selective pressure. A green fluorescent protein gene (gfp) from pHT315-gfp was successfully cloned and expressed in A. hydrophila strain X2 using pAEsv-1, and 82.3% ± 2.5% of cells maintained the recombinant plasmid after one week in liquid culture without kanamycin. These results suggested that pAEsv-1 might potentially be used as a stable cloning vector for A. hydrophila, which might facilitate genetic studies of A. hydrophila.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"108 ","pages":"Article 102490"},"PeriodicalIF":2.6,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102490","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37598326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

SGI0, a relative of Salmonella genomic islands SGI1 and SGI2, lacking a class 1 integron, found in Proteus mirabilis SGI0是沙门氏菌基因组岛SGI1和SGI2的近亲，缺乏1类整合子，发现于变形杆菌中

IF 2.6 4区生物学

Plasmid Pub Date : 2020-01-01 DOI: 10.1016/j.plasmid.2019.102453

Claire de Curraize , Eliane Siebor , Catherine Neuwirth , Ruth M. Hall

{"title":"SGI0, a relative of Salmonella genomic islands SGI1 and SGI2, lacking a class 1 integron, found in Proteus mirabilis","authors":"Claire de Curraize , Eliane Siebor , Catherine Neuwirth , Ruth M. Hall","doi":"10.1016/j.plasmid.2019.102453","DOIUrl":"10.1016/j.plasmid.2019.102453","url":null,"abstract":"<div>Several groups of integrative mobilizable elements (IMEs) that harbour a class 1 integron carrying antibiotic resistance genes have been found at the 3′-end of the chromosomal trmE gene. Here, a new IME, designated SGI0, was found in trmE in the sequenced and assembled genome of a French clinical, multiply antibiotic resistant Proteus mirabilis strain, Pm1LENAR. SGI0 shares the same gene content as the backbones of SGI1 and SGI2 (overall 97.6% and 97.7% nucleotide identity, respectively) but it lacks a class 1 integron. However, SGI0 is a mosaic made up of segments with >98.5% identity to SGI1 and SGI2 interspersed with segments sharing 74–95% identity indicating that further diverged backbone types exist and that recombination between them is occurring. The structure of SGI1-V, here re-named SGI-V, which lacks two SGI1 (S023 and S024) backbone genes and includes a group of additional genes in the backbone, was re-examined. In regions shared with SGI1, the backbones shared 97.3% overall identity with the differences distributed in patches with various levels of identity. The class 1 integron is also in a slightly different position with the target site duplication AAATT instead of ACTTG for SGI1 and variants, indicating that it was acquired independently. The Pm1LENAR resistance genes are in the chromosome, in Tn7 and an ISEcp1-mobilised segment.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"107 ","pages":"Article 102453"},"PeriodicalIF":2.6,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2019.102453","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45699341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

Corrigendum to “De novo synthesis of the complete genome of coxsackievirus A10 based on Golden Gate cloning combined with promoter reconstruction” [Plasmid 103 (2019) 17–24] “基于金门克隆结合启动子重建的柯萨奇病毒A10全基因组从头合成”的勘误表[质粒103 (2019)17-24]

IF 2.6 4区生物学

Plasmid Pub Date : 2020-01-01 DOI: 10.1016/j.plasmid.2019.102439

Meng Li , Jing Chen

引用次数: 0

The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance 产气荚膜梭菌的Tcp质粒需要有resP基因才能保证稳定的遗传

IF 2.6 4区生物学

Plasmid Pub Date : 2020-01-01 DOI: 10.1016/j.plasmid.2019.102461

Sarah Revitt-Mills , Carmen Lao , Marie Archambault , Dena Lyras , Julian I. Rood , Vicki Adams

{"title":"The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance","authors":"Sarah Revitt-Mills , Carmen Lao , Marie Archambault , Dena Lyras , Julian I. Rood , Vicki Adams","doi":"10.1016/j.plasmid.2019.102461","DOIUrl":"10.1016/j.plasmid.2019.102461","url":null,"abstract":"<div>Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"107 ","pages":"Article 102461"},"PeriodicalIF":2.6,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2019.102461","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42661560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

A novel expression vector for Corynebacterium glutamicum with an auxotrophy complementation system 谷氨酸棒状杆菌具有营养缺失互补系统的新型表达载体

IF 2.6 4区生物学

Plasmid Pub Date : 2020-01-01 DOI: 10.1016/j.plasmid.2019.102476

Ying Li , Yuqing Ai , Junzheng Zhang , Jingxuan Fei , Bingnan Liu , Jing Wang , Meng Li , Qiancheng Zhao , Jinzhu Song

{"title":"A novel expression vector for Corynebacterium glutamicum with an auxotrophy complementation system","authors":"Ying Li , Yuqing Ai , Junzheng Zhang , Jingxuan Fei , Bingnan Liu , Jing Wang , Meng Li , Qiancheng Zhao , Jinzhu Song","doi":"10.1016/j.plasmid.2019.102476","DOIUrl":"10.1016/j.plasmid.2019.102476","url":null,"abstract":"<div>Corynebacterium glutamicum is an important industrial strain used for the production of amino acids and vitamins. Most tools developed for overexpression of genes in C. glutamicum are based on the inducible promoter regulated by the lacIq gene or contain an antibiotic resistance gene as a selection marker. These vectors are essential for rapid identification of recombinant strains and detailed study of gene functions, but, as a considerable disadvantage, these vectors are not suitable for large-scale industrial production due to the need for the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) and antibiotics. In this study, the novel Escherichia coli-C. glutamicum shuttle expression vector pLY-4, derived from the expression vector pXMJ19, was constructed. The constitutive vector pLY-4 contains a large multiple cloning site, the strong promoter tacM and two selective markers: the original chloramphenicol resistance gene cat is used for molecular cloning operations, and the auxotrophy complementation marker alr, which can be stably replicated in the auxotrophic host strain without antibiotic selection pressure, is used for industrial fermentation. Heterologous expression of the gapC gene using the vector pLY-4 in C. glutamicum for L-methionine production indicated the potential application of pLY-4 in the development of C. glutamicum strain engineering and industrial fermentation.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"107 ","pages":"Article 102476"},"PeriodicalIF":2.6,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2019.102476","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54892750","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8

Two novel transcriptional reporter systems for monitoring Helicobacter pylori stress responses 监测幽门螺杆菌应激反应的两种新型转录报告系统

IF 2.6 4区生物学

Plasmid Pub Date : 2019-11-01 DOI: 10.1016/j.plasmid.2019.102442

A.M. Belova , D.V. Basmanov , V.V. Babenko , O.V. Podgorny , T.V. Mitko , K.A. Prusakov , D.V. Klinov , V.N. Lazarev

{"title":"Two novel transcriptional reporter systems for monitoring Helicobacter pylori stress responses","authors":"A.M. Belova , D.V. Basmanov , V.V. Babenko , O.V. Podgorny , T.V. Mitko , K.A. Prusakov , D.V. Klinov , V.N. Lazarev","doi":"10.1016/j.plasmid.2019.102442","DOIUrl":"10.1016/j.plasmid.2019.102442","url":null,"abstract":"<div>Helicobacter pylori, a human pathogen linked to many stomach diseases, is well adapted to colonize aggressive gastric environments, and its virulence factors contribute this adaptation. Here, we report the construction of two novel H. pylori vectors, pSv2 and pSv4, carrying a reporter gene fused to the promoters of virulence factor genes for monitoring the response of single H. pylori cells to various stresses. H. pylori cryptic plasmids were modified by the introduction of the Escherichia coli origin of replication, chloramphenicol resistance cassette, and promoterless gfp gene to produce E. coli/H. pylori shuttle vectors. The promoter regions of vacA and ureA genes encoding well-characterized H. pylori virulence factors were fused to the promoterless gfp gene. Recording the GFP fluorescence signal from the genetically modified H. pylori cells immobilized in specifically designed microfluidic devices revealed the response of transcriptional reporter systems to osmotic stress, acidic stress, elevated Ni2+ concentration or iron chelation. Our observations validate the utility of the pSv2 and pSv4 vectors to monitor the regulation of virulence factor genes in diverse strains and clinical isolates of H. pylori.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"106 ","pages":"Article 102442"},"PeriodicalIF":2.6,"publicationDate":"2019-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2019.102442","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48044333","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Plasmid pSfr64a and the symbiotic plasmid pSfr64b of Sinorhizobium fredii GR64 control each other's conjugative transfer through quorum-sensing elements fredii Sinorhizobium GR64的质粒pSfr64a和共生质粒pSfr64b通过群体感应元件相互控制对方的共轭转移。

IF 2.6 4区生物学

Plasmid Pub Date : 2019-11-01 DOI: 10.1016/j.plasmid.2019.102443

Laura Cervantes , Fabiola Miranda-Sánchez , Gonzalo Torres Tejerizo , David Romero , Susana Brom

{"title":"Plasmid pSfr64a and the symbiotic plasmid pSfr64b of Sinorhizobium fredii GR64 control each other's conjugative transfer through quorum-sensing elements","authors":"Laura Cervantes , Fabiola Miranda-Sánchez , Gonzalo Torres Tejerizo , David Romero , Susana Brom","doi":"10.1016/j.plasmid.2019.102443","DOIUrl":"10.1016/j.plasmid.2019.102443","url":null,"abstract":"<div>Rhizobia are nitrogen-fixing symbionts of plants. Their genomes frequently contain large plasmids, some of which are able to perform conjugative transfer. Plasmid pSfr64a from Sinorhizobium fredii GR64 is a conjugative plasmid, whose transfer is regulated by quorum sensing genes encoded by itself (traR64a, traI64a), in the symbiotic plasmid pSfr64b (traR64b, traI64b), and in the chromosome (ngrI). Also, transfer of pSfr64b requires quorum sensing elements encoded in this plasmid (traR64b, traI64b), in pSfr64a (traR64a), and in the chromosome (ngrI). These results demonstrate that pSfr64a and the symbiotic plasmid depend on each other for conjugative transfer. Plasmid pSfr64a from S. fredii GR64 is unable to transfer from the genomic background of Rhizobium etli CFN42. Our results show that the relaxase of pRet42a is able to process the oriT of pSfr64a, and viceversa, underlining their functional similarity and suggesting that in addition to the external signals, the “cytoplasmic environment” may pose a barrier to plasmid dissemination, even if the plasmids are functional in other aspects.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"106 ","pages":"Article 102443"},"PeriodicalIF":2.6,"publicationDate":"2019-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2019.102443","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47515571","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Synthetic promoter for efficient and muscle-specific expression of exogenous genes 外源基因高效和肌肉特异性表达的合成启动子

IF 2.6 4区生物学

Plasmid Pub Date : 2019-11-01 DOI: 10.1016/j.plasmid.2019.102441

Yili Liu , Yutong He , Yong Wang , Ming Liu , Mingfeng Jiang , Rong Gao , Gang Wang

{"title":"Synthetic promoter for efficient and muscle-specific expression of exogenous genes","authors":"Yili Liu , Yutong He , Yong Wang , Ming Liu , Mingfeng Jiang , Rong Gao , Gang Wang","doi":"10.1016/j.plasmid.2019.102441","DOIUrl":"10.1016/j.plasmid.2019.102441","url":null,"abstract":"<div>Synthetic promoters (SPs) have many advantages over their natural counterparts, especially with regard to transcriptional activity and tissue specificity. Here, we report a new strategy to construct SPs for efficient and muscle-specific gene expression. First, 19 nucleic acid motifs classified to 3 kinds of transcriptional regulatory elements were rationally selected. A recombinant promoter library was constructed by randomly assembling these motifs. Second, the transcriptional activities of ~1200 SPs were screened by intramuscular expression of several reporter genes in different cell lines for activity higher than that of the cytomegalovirus (CMV) promoter, with SP-301 finally identified as the strongest. A single intramuscular injection of mice with an SP-301 plasmid expressing mouse growth hormone releasing hormone accelerated mouse growth significantly over 24 days. Third, the muscle specificity of SP-301 was confirmed in transgenic mice. Finally, in comparison with the CMV promoter, SP-301 accelerated translocation and increased the level of plasmid in the nuclei of myoblast cells to a greater extent than in non-muscle cells. Altogether, the study has provided a more rational strategy to construct efficient and tissue-specific promoters, with the promoter SP-301 exhibiting promising potential for establishing an intramuscular gene expression system for therapeutic applications.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"106 ","pages":"Article 102441"},"PeriodicalIF":2.6,"publicationDate":"2019-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2019.102441","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42716271","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Dissemination of blaKPC-2 in an NTEKPC by an IncX5 plasmid 用IncX5质粒在NTEKPC中传播blaKPC-2

IF 2.6 4区生物学

Plasmid Pub Date : 2019-11-01 DOI: 10.1016/j.plasmid.2019.102446

Rosineide Cardoso de Souza , Andrei Nicoli Gebieluca Dabul , Camila Maria dos Santos Boralli , Luíza Zuvanov , Ilana Lopes Baratella da Cunha Camargo

{"title":"Dissemination of blaKPC-2 in an NTEKPC by an IncX5 plasmid","authors":"Rosineide Cardoso de Souza , Andrei Nicoli Gebieluca Dabul , Camila Maria dos Santos Boralli , Luíza Zuvanov , Ilana Lopes Baratella da Cunha Camargo","doi":"10.1016/j.plasmid.2019.102446","DOIUrl":"10.1016/j.plasmid.2019.102446","url":null,"abstract":"<div>blaKPC-2 is disseminated worldwide usually in Tn4401, a Tn3-family transposon, and primarily in Klebsiella pneumoniae ST258, a well-known lineage that is distributed worldwide and responsible for several outbreaks. Although occurring rarely, blaKPC-2 has been described in non-Tn4401 elements (NTEKPCs), first in China and then in a few other countries. This study reports the dissemination of a blaKPC-2-carrying NTEKPC among ST11/CG258 K. pneumoniae strains and ST1642 K. quasipneumoniae subsp. quasipneumoniae AMKP9 in an Amazonian hospital. The dissemination was due to pAMKP10, an ~48 kbp IncX5 plasmid carrying ΔISKpn6/blaKPC-2/ISKpn27 in a Tn1722-based unit. Although similar to NTEKPC-Ia from pKP048 described in China, a different transposase is present upstream of ISKpn27. Additionally, mutations were identified downstream of ISKpn27 but did not affect the blaKPC-2 promoter regions. pAMKP10 conjugated in vitro only from CG258 isolates. Since CG258 strains are generally well adapted to the hospital environment, it is significant that pAMKP10 has found its way into this clinically significant clonal group. The impact of inter- and intraspecies dissemination of NTEKPCs and IncX5 plasmids harboring carbapenem resistance genes is unknown, but monitoring these plasmids could reveal their dissemination preferences.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"106 ","pages":"Article 102446"},"PeriodicalIF":2.6,"publicationDate":"2019-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2019.102446","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42861912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Seamless insert-plasmid assembly at sub-terminal homologous sequences 亚末端同源序列的无缝插入-质粒组装

IF 2.6 4区生物学

Plasmid Pub Date : 2019-11-01 DOI: 10.1016/j.plasmid.2019.102445

Anna-Sophia Krebs , Tobias Bierig , Gabriella Collu , Roger M. Benoit

{"title":"Seamless insert-plasmid assembly at sub-terminal homologous sequences","authors":"Anna-Sophia Krebs , Tobias Bierig , Gabriella Collu , Roger M. Benoit","doi":"10.1016/j.plasmid.2019.102445","DOIUrl":"10.1016/j.plasmid.2019.102445","url":null,"abstract":"<div>The engineering of fusion proteins for structural biology and protein nanotechnology often requires seamless DNA assembly with slight variations in the domain boundaries. To improve the molecular biology workflow for such projects, we evaluated the use of sub-terminal homologous sequences (HS) for co-transformation cloning and for T5 exonuclease / Phusion DNA polymerase mediated in vitro assembly. To quantify the effects of different HS-to-ends distances on cloning efficiency, we designed a blue-white-pink screening system that allowed us to easily identify positive clones (blue colonies), negative clones resulting from circular template plasmid (pink colonies) and negative colonies originating from linearized plasmids that have recircularized without an insert (white colonies). Our experiments show that both methods are feasible with HS-to-ends distances up to at least 10 base pairs. Using a combination of co-transformation cloning at sub-terminal HS and nucleotide insertions in non-annealing primer 5′-overhangs, we integrated a fusion protein into the third intracellular loop (ICL) of a G-protein-coupled receptor (GPCR) with nine different linker boundaries, using only a single plasmid linearization reaction. This molecular cloning approach is an invaluable tool for protein engineering, protein nanotechnology and synthetic biology that extends the range of applications of DNA assembly strategies.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"106 ","pages":"Article 102445"},"PeriodicalIF":2.6,"publicationDate":"2019-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2019.102445","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45463865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1