IF 2.6 4区生物学

Plasmid Pub Date : 2020-09-01 DOI: 10.1016/j.plasmid.2020.102530

Christopher J. Harmer, Carol H. Pong, Ruth M. Hall

{"title":"Structures bounded by directly-oriented members of the IS26 family are pseudo-compound transposons.","authors":"Christopher J. Harmer, Carol H. Pong, Ruth M. Hall","doi":"10.1016/j.plasmid.2020.102530","DOIUrl":"10.1016/j.plasmid.2020.102530","url":null,"abstract":"<div>Antibiotic resistance genes are often found in structures bounded by copies of IS26, IS257/IS431 or IS1216 that resemble compound (or composite) transposons. However, because of the mechanisms used by IS26 family members, namely that they form cointegrates but cannot resolve them, none of these structures can move together as a coherent single unit. Apparent transposition of these structures is possible via a 2-step process but only if the IS are in direct orientation. An intermolecular reaction catalysed by the IS-encoded transposase and an intramolecular homologous recombination step can occur in either order. In one route, one of the IS bounding the structure forms a cointegrate between the DNA molecule carrying it and a target molecule. Cointegrates formed by either copy-in or targeted conservative routes contain three directly-oriented IS copies and can be resolved by homologous recombination between specific pairs of IS, with one pair leading to apparent transposition of the whole structure. In the other route, homologous recombination first forms a circular intermediate, a translocatable unit or TU, which is incorporated by the transposase either at a random site or adjacent to another IS copy in a target molecule. We therefore conclude that the transposon-like structures are not compound (or composite) transposons and the nomenclature for them should be revised. We propose that the term “pseudo compound transposon” (PCT), first coined in 1989, should be used to describe those structures where the IS are in direct orientation. Structures with the IS in opposite orientation should not be named as transposons.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"111 ","pages":"Article 102530"},"PeriodicalIF":2.6,"publicationDate":"2020-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102530","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38331613","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: 43

Novel conditional plasmids regulated by chemical switches provide versatile tools for genetic engineering in Escherichia coli 由化学开关调节的新型条件质粒为大肠杆菌的基因工程提供了多功能工具

IF 2.6 4区生物学

Plasmid Pub Date : 2020-09-01 DOI: 10.1016/j.plasmid.2020.102531

André Riedl , Simone Gruber , Zsolt Ruzsics

{"title":"Novel conditional plasmids regulated by chemical switches provide versatile tools for genetic engineering in Escherichia coli","authors":"André Riedl , Simone Gruber , Zsolt Ruzsics","doi":"10.1016/j.plasmid.2020.102531","DOIUrl":"10.1016/j.plasmid.2020.102531","url":null,"abstract":"<div>Engineering bacterial genomes or foreign DNA cloned as bacterial artificial chromosomes (BACs) relies on usage of helper plasmids, which deliver the desired tools transiently into the bacteria to be modified. After the anticipated action is completed the helper plasmids need to be cured. To make this efficient, plasmids are used that are maintained by conditional amplicons or carry a counter-selection marker. Here, we describe new conditional plasmids that can be maintained or cured by using chemical induction or repression. Our method is based on the dependency of plasmids carrying ori6Kγ origin of replication on the presence of protein Π. Ori6Kγ based plasmids are tightly regulated conditional constructs, but they require usually special E. coli strains to operate. To avoid this, we placed the Π protein expression under the control of a co-expressed conditional repressor. Regulating the maintenance of plasmids with administration or removal of chemicals is fully compatible with any other conditional amplicons applied to date. Here, we describe methods for inducing sites specific recombination of BACs as an example. However, the same strategy might be used to construct appropriate helper plasmids for any other transient components of genome editing methodologies such as λred recombinases or CRISPR/Cas components.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"111 ","pages":"Article 102531"},"PeriodicalIF":2.6,"publicationDate":"2020-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102531","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38371741","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

Formation of new PHE plasmids in pseudomonads in a phenol-polluted environment 苯酚污染环境中假单胞菌新PHE质粒的形成

IF 2.6 4区生物学

Plasmid Pub Date : 2020-07-01 DOI: 10.1016/j.plasmid.2020.102504

Eve Elken, Eeva Heinaru, Merike Jõesaar, Ain Heinaru

{"title":"Formation of new PHE plasmids in pseudomonads in a phenol-polluted environment","authors":"Eve Elken, Eeva Heinaru, Merike Jõesaar, Ain Heinaru","doi":"10.1016/j.plasmid.2020.102504","DOIUrl":"10.1016/j.plasmid.2020.102504","url":null,"abstract":"<div>Several years ago, a laboratory-constructed plasmid with a single-component phenol monooxygenase gene (pheBA operon) flanked by two IS elements was released to a phenol-polluted area. During the following years, we found in the test area widely distributed pheBA operon-containing bacteria. The new pheBA+ strains belong predominantly to the Pseudomonas fluorescens group, and they did not arise via selection of the released PHE plasmid. On the contrary, the formation of several different types of PHE plasmids occurred, namely pPHE101 (60,958 bp) from the IncP-9 group, non-transferable plasmid pPHE69 (44,717 bp), mobilizable plasmid pPHE20 (39,609 bp) and the IncP-7 type plasmid pPHE24ΔpheBA (120,754 bp), in which the pheBA operon was translocated from the plasmid to the chromosome. In two cases, PHE plasmid-bearing strains exist in a multi-plasmid state, also containing the non-catabolic plasmids pG20 (133,709 bp) and pG69 (144,433 bp) with backbones sharing 97% DNA identity and with redundant genes for the initiation of replication, repA1and repA2, of which only one was active. Seemingly, several other plasmids and bacterial features besides the pheBA operon were involved in selective distribution of catabolic operons in the natural environment. The comparison of the genetic structure of plasmids and IS elements' functions, as well as resistance to heavy metals of seven completely sequenced plasmids, are discussed.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"110 ","pages":"Article 102504"},"PeriodicalIF":2.6,"publicationDate":"2020-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102504","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37832659","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: 3

Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida 恶臭假单胞菌基因(过)表达双诱导双表达体系的构建

IF 2.6 4区生物学

Plasmid Pub Date : 2020-07-01 DOI: 10.1016/j.plasmid.2020.102514

Rahul Gauttam , Aindrila Mukhopadhyay , Steven W. Singer

{"title":"Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida","authors":"Rahul Gauttam , Aindrila Mukhopadhyay , Steven W. Singer","doi":"10.1016/j.plasmid.2020.102514","DOIUrl":"10.1016/j.plasmid.2020.102514","url":null,"abstract":"<div>Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual-inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modified for use in P. putida. This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida. Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters Ptac or PtetR/tetA. Functional validation of pRGPDuo2 was confirmed by the co-expression of genes for the fluorescent proteins namely, superfolder green fluorescent protein (sfGFP), and red fluorescent protein (RFP). Moreover, the strength of the fluorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression profiling and adds to the tools available for P. putida metabolic engineering.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"110 ","pages":"Article 102514"},"PeriodicalIF":2.6,"publicationDate":"2020-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102514","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38015352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 13

Construction of a novel CRISPRi-based tool for silencing of multiple genes in Mycobacterium tuberculosis 构建一种基于crispr的新型结核分枝杆菌多基因沉默工具

IF 2.6 4区生物学

Plasmid Pub Date : 2020-07-01 DOI: 10.1016/j.plasmid.2020.102515

Nisheeth Agarwal

{"title":"Construction of a novel CRISPRi-based tool for silencing of multiple genes in Mycobacterium tuberculosis","authors":"Nisheeth Agarwal","doi":"10.1016/j.plasmid.2020.102515","DOIUrl":"10.1016/j.plasmid.2020.102515","url":null,"abstract":"<div>Due to lipid-rich cell wall, slow growth and pathogenic nature, it is difficult to manipulate Mycobacterium tuberculosis (Mtb) genome by conventional tools. Recently we have introduced a novel CRISPRi approach for repression of genes in mycobacteria. Although the existing CRISPRi plasmid is proven useful for silencing individual targets, disruption of multiple ORFs remains challenging in mycobacteria. Herein, we report construction of the guide sequence expressing plasmid, pGrna to facilitate cloning and expression of multiple guide sequence cassettes targeting a versatile set of Mtb genes from a single plasmid. Using the modified plasmid, pGrna2, it was shown that expression of all the 10 extracellular sigma factor-encoding genes together with sigB and sigF can be efficiently repressed in Mtb expressing dCas9. In vitro growth analysis indicates that simultaneous knockdown of these non-essential transcriptional regulators is lethal for growth. Importantly, the Δ12sig strain exhibits sensitivity to transcriptional inhibitor rifampicin and oxidative stress diamide, further implying involvement of these genes in controlling bacterial stress response. To the best of my knowledge, this is the first report wherein 12 genes have been efficiently silenced together in a single recombinant strain of Mtb. The modified pGrna2 plasmid offers a powerful tool to decipher the functioning of genes that are redundant or regulate a particular metabolic pathway and can be useful in identification of novel anti-tuberculosis drug targets.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"110 ","pages":"Article 102515"},"PeriodicalIF":2.6,"publicationDate":"2020-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102515","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38041995","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: 9

Plasmidome of an environmental Acinetobacter lwoffii strain originating from a former gold and arsenic mine 原金砷矿环境不动杆菌lwoffii菌株的质粒

IF 2.6 4区生物学

Plasmid Pub Date : 2020-07-01 DOI: 10.1016/j.plasmid.2020.102505

Tomasz Walter, Joanna Klim, Marcin Jurkowski, Jan Gawor, Iwona Köhling, Małgorzata Słodownik, Urszula Zielenkiewicz

{"title":"Plasmidome of an environmental Acinetobacter lwoffii strain originating from a former gold and arsenic mine","authors":"Tomasz Walter, Joanna Klim, Marcin Jurkowski, Jan Gawor, Iwona Köhling, Małgorzata Słodownik, Urszula Zielenkiewicz","doi":"10.1016/j.plasmid.2020.102505","DOIUrl":"10.1016/j.plasmid.2020.102505","url":null,"abstract":"<div>Emerging important Acinetobacter strains commonly accommodate a plethora of mobile elements including plasmids of different size. Plasmids, apart from encoding modules enabling their self-replication and/or transmission, can carry a diverse number of genes, allowing the host cell to survive in an environment that would otherwise be lethal or restrictive for growth. The present study characterizes the plasmidome generated from an arsenic-resistant strain named ZS207, classified as Acinetobacter lwoffii. Sequencing effort revealed the presence of nine plasmids in the size between 4.3 and 38.4 kb as well as one 186.6 kb megaplasmid. All plasmids, except the megaplasmid, do apparently not confer distinguishing phenotypic features. In contrast, the megaplasmid carries arsenic and heavy metals resistance regions similar to those found in permafrost A. lwoffii strains. In-depth in silico analyses have shown a significant similarity between the regions from these plasmids, especially concerning multiple transposable elements, transfer and mobilization genes, and toxin-antitoxin systems.Since ars genes encode proteins of major significance in terms of potential use in bioremediation, arsenic resistance level of ZS207 was determined and the functionality of selected ars genes was examined. Additionally, we checked the functionality of plasmid-encoded toxin-antitoxin systems and their impact on the formation of persister cells.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"110 ","pages":"Article 102505"},"PeriodicalIF":2.6,"publicationDate":"2020-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102505","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37910309","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8

The cell-penetrating YopM protein-functionalized quantum dot-plasmid DNA conjugate as a novel gene delivery vector 细胞穿透YopM蛋白功能化量子点-质粒DNA偶联物作为一种新的基因传递载体

IF 2.6 4区生物学

Plasmid Pub Date : 2020-07-01 DOI: 10.1016/j.plasmid.2020.102513

Özge Uğurlu , Fırat Barış Barlas , Serap Evran , Suna Timur

{"title":"The cell-penetrating YopM protein-functionalized quantum dot-plasmid DNA conjugate as a novel gene delivery vector","authors":"Özge Uğurlu , Fırat Barış Barlas , Serap Evran , Suna Timur","doi":"10.1016/j.plasmid.2020.102513","DOIUrl":"10.1016/j.plasmid.2020.102513","url":null,"abstract":"<div>Non-viral gene delivery systems have great potential for safe and efficient gene therapy, while inefficient cellular and nuclear uptake remain as the major hurdles. Novel approaches are needed to enhance the transfection efficiency of non-viral vectors. In accordance with this need, the objective of this study was to construct a non-viral vector that could achieve gene delivery without using additional lipid-based transfection agent. We aimed to impart self-delivery property to a non-viral vector by using the cell and nucleus penetrating properties of YopM proteins from the three Yersinia spp. (Y. pestis, Y. enterocolotica and Y. pseudotuberculosis). Plasmid DNA (pDNA) encoding green fluorescent protein (GFP) was labeled with quantum dots (QDs) via peptide-nucleic acid (PNA) recognition site. Recombinant YopM protein was then attached to the conjugate via a second PNA recognition site. The YopM ̶ QDs ̶ pDNA conjugate was transfected into HeLa cells without using additional transfection reagent. All three conjugates produced GFP fluorescence, indicating that the plasmid was successfully delivered to the nucleus. As control, naked pDNA was transfected into the cells by using a commercial transfection reagent. The Y. pseudotuberculosis YopM-functionalized conjugate achieved the highest GFP expression, compared to other two YopM proteins and the transfection reagent. To the best of our knowledge, YopM protein was used for the first time in a non-viral gene delivery vector.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"110 ","pages":"Article 102513"},"PeriodicalIF":2.6,"publicationDate":"2020-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102513","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38013236","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

Incompatibility and phylogenetic relationship of I-complex plasmids i -复合体质粒的不亲和性及其系统发育关系

IF 2.6 4区生物学

Plasmid Pub Date : 2020-05-01 DOI: 10.1016/j.plasmid.2020.102502

Marta Rozwandowicz , Joost Hordijk , Alex Bossers , Aldert L. Zomer , Jaap A. Wagenaar , Dik J. Mevius , Michael S.M. Brouwer

{"title":"Incompatibility and phylogenetic relationship of I-complex plasmids","authors":"Marta Rozwandowicz , Joost Hordijk , Alex Bossers , Aldert L. Zomer , Jaap A. Wagenaar , Dik J. Mevius , Michael S.M. Brouwer","doi":"10.1016/j.plasmid.2020.102502","DOIUrl":"10.1016/j.plasmid.2020.102502","url":null,"abstract":"<div>Plasmid incompatibility is the inability of two plasmids to be stably maintained in one cell, resulting in loss of one of the plasmids in daughter cells. Dislodgement is a phenotypically distinct form of incompatibility, described as an imperfect reproduction, manifesting in rapid exclusion of a resident plasmid after superinfection. The relationship between plasmids of the phenotypic incompatibility groups IncB/O and IncZ is unclear. Their inability to co-exist was initially referred to as dislodgement while other research reached the conclusion that IncB/O and IncZ plasmids are incompatible. In this manuscript we re-evaluated the relationship between IncB/O and IncZ plasmids to settle these conflicting conclusions. We performed dislodgement testing of R16Δ (IncB/O) and pSFE-059 (IncZ) plasmids by electroporation in a bacterial cell and checked their stability. Stability tests of the obtained plasmid pair showed that the IncB/O plasmid was exclusively and almost completely lost from the heteroplasmid Escherichia coli population. Other IncB/O – IncZ pairs could not form a heteroplasmid population, using conjugation or electroporation. Our data supports the previous suggestion that IncB/O and IncZ plasmids may be considered phenotypically incompatible.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"109 ","pages":"Article 102502"},"PeriodicalIF":2.6,"publicationDate":"2020-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102502","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37738324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Tailor-made sRNAs: a plasmid tool to control the expression of target mRNAs in Pseudomonas putida 定制的sRNAs:一种控制恶臭假单胞菌靶mrna表达的质粒工具

IF 2.6 4区生物学

Plasmid Pub Date : 2020-05-01 DOI: 10.1016/j.plasmid.2020.102503

Patrícia Apura , Margarida Saramago , Alexandra Peregrina , Sandra C. Viegas , Sandra M. Carvalho , Lígia M. Saraiva , Cecília M. Arraiano , Susana Domingues

{"title":"Tailor-made sRNAs: a plasmid tool to control the expression of target mRNAs in Pseudomonas putida","authors":"Patrícia Apura , Margarida Saramago , Alexandra Peregrina , Sandra C. Viegas , Sandra M. Carvalho , Lígia M. Saraiva , Cecília M. Arraiano , Susana Domingues","doi":"10.1016/j.plasmid.2020.102503","DOIUrl":"10.1016/j.plasmid.2020.102503","url":null,"abstract":"<div>Pseudomonas putida is a highly attractive production system for industrial needs. However, for its improvement as a biocatalyst at the industrial level, modulation of its gene expression is urgently needed. We report the construction of a plasmid expressing a small RNA-based system with the potential to be used for different purposes. Due to the small RNAs modular composition, the design facilities and ability to tune gene expression, they constitute a powerful tool in genetic and metabolic engineering. In the tool presented here, customized sRNAs are expressed from a plasmid and specifically directed to any region of a chosen target. Expression of these customized sRNAs is shown to differentially modulate the level of endogenous and heterologous reporter genes. The antisense interaction of the sRNA with the mRNA produces different outcomes. Depending on the particularity of each sRNA-target mRNA pair, we demonstrate the duality of this system, which is able either to decrease or increase the expression of the same given gene. This system combines high specificity with the potential to be widely applied, due to its predicted ability to modulate the expression of virtually any given gene. This plasmid can be used to redesign P. putida metabolism, fulfilling an important industrial gap.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"109 ","pages":"Article 102503"},"PeriodicalIF":2.6,"publicationDate":"2020-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102503","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37768411","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: 14

Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli 基于强合成固定相启动子的大肠杆菌基因表达体系

IF 2.6 4区生物学

Plasmid Pub Date : 2020-05-01 DOI: 10.1016/j.plasmid.2020.102491

Jananee Jaishankar, Preeti Srivastava

{"title":"Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli","authors":"Jananee Jaishankar, Preeti Srivastava","doi":"10.1016/j.plasmid.2020.102491","DOIUrl":"10.1016/j.plasmid.2020.102491","url":null,"abstract":"<div>The Gram-negative bacterium Escherichia coli has been the work horse for recombinant protein production since the past several years. However, most of the gene expression systems used either require expensive inducers or exhibit low strength. In the present study, we have generated a strong promoter by repeated rounds of random mutagenesis in a stationary phase promoter isolated from Gordonia sp. IITR100. The promoter activity increased 16-fold as compared to the wild-type promoter. The resultant synthetic promoter showed β-galactosidase activities of ~16,000 Miller units which is comparable to the strong T7 promoter ~13,000 Miller units. The amount of LacZ produced by the synthetic promoter was found to be active for several days in stationary phase. The advantage of this synthetic promoter over T7 promoter includes its stationary phase auto-inducibility thereby saving the cost of addition of inducers. Expression of GFPuv was observed in all the cells of E. coli due to the absence of requirement of inducer. A general-purpose vector containing the synthetic promoter with an MCS ready for use has been developed in the study. It has also been used to demonstrate the production of two heterologous proteins.</div>","PeriodicalId":49689,"journal":{"name":"Plasmid","volume":"109 ","pages":"Article 102491"},"PeriodicalIF":2.6,"publicationDate":"2020-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.plasmid.2020.102491","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37623061","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

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