scholarly journals Engineering a minimal cloning vector from a pUC18 plasmid backbone with an extended multiple cloning site

BioTechniques ◽  
2019 ◽  
Vol 66 (6) ◽  
pp. 254-259 ◽  
Author(s):  
Jens Staal ◽  
Kübra Alci ◽  
Wouter De Schamphelaire ◽  
Martine Vanhoucke ◽  
Rudi Beyaert
Keyword(s):  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Plasmid Backbone

scholarly journals Engineering a Minimal Cloning Vector from a pUC18 Plasmid Backbone with an Extended Multiple Cloning Site

2018 ◽  
Author(s):  
Jens Staal ◽  
Wouter De Schamphelaire ◽  
Rudi Beyaert
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
Restriction Enzyme ◽  
Essential Role ◽  
Building Blocks ◽  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Unique Restriction ◽  
Plasmid Backbone

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

scholarly journals Engineering a Minimal 1185 Bp Cloning Vector from a Puc18 Plasmid Backbone with an Extended Multiple Cloning Site

2018 ◽  
Author(s):  
Jens Staal ◽  
Wouter De Schamphelaire ◽  
Rudi Beyaert
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
Restriction Enzyme ◽  
Essential Role ◽  
Building Blocks ◽  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Unique Restriction ◽  
Plasmid Backbone

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of a ~1kb fully functional cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

scholarly journals Sequence Analysis of Two Cryptic Plasmids from Bifidobacterium longum DJO10A and Construction of a Shuttle Cloning Vector

2006 ◽  
Vol 72 (1) ◽  
pp. 527-535 ◽  
Author(s):  
Ju-Hoon Lee ◽  
Daniel J. O’Sullivan
Keyword(s):  
Recombination Event ◽  
Sequence Similarity ◽  
Bifidobacterium Longum ◽  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Rhodococcus Rhodochrous ◽  
Gram Positive ◽  
High Sequence Identity ◽  
Chloramphenicol Resistance ◽  
E Coli

ABSTRACT Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.

scholarly journals Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

2007 ◽  
Vol 73 (23) ◽  
pp. 7542-7547 ◽  
Author(s):  
Dag Anders Brede ◽  
Sheba Lothe ◽  
Zhian Salehian ◽  
Therese Faye ◽  
Ingolf F. Nes
Keyword(s):  
Selection Marker ◽  
Multiple Cloning Site ◽  
Propionibacterium Freudenreichii ◽  
Expression Plasmid ◽  
Physiochemical Properties ◽  
Food Grade ◽  
Cloning System ◽  
Immunity Gene ◽  
Membrane Bound ◽  
High Level

ABSTRACT This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive PpampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ∼91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

The complete nucleotide sequences of the SacBII Kan Domain of the P1 pAD10-SacBII cloning vector and three cosmid cloning vectors: pTCF, svPHEP, and LAWRIST16

1994 ◽  
Vol 11 (5-6) ◽  
pp. 181-186 ◽  
Author(s):  
Hua-Qin Pan ◽  
Ying-Ping Wang ◽  
Stephanie L. Chissoe ◽  
Angelika Bodenteich ◽  
Zhili Wang ◽  
...  
Keyword(s):  
Nucleotide Sequences ◽  
Cloning Vector ◽  
Cloning Vectors ◽  
Cosmid Cloning

scholarly journals The doses of plasmid backbone plays a major role in determining the HBV clearance in hydrodynamic injection mouse model

2018 ◽  
Vol 15 (1) ◽  
Author(s):  
Xian Wang ◽  
Jianmin Zhu ◽  
Yong Zhang ◽  
Yue Li ◽  
Tai Ma ◽  
...  
Keyword(s):  
Mouse Model ◽  
Hydrodynamic Injection ◽  
Plasmid Backbone

scholarly journals Characterization of a Laboratory-Derived, High-Level Ampicillin-Resistant Salmonella enterica Serovar Typhimurium Strain That Caused Meningitis in an Infant

2002 ◽  
Vol 46 (5) ◽  
pp. 1604-1606 ◽  
Author(s):  
Cheng-Hsun Chiu ◽  
Chishih Chu ◽  
Lin-Hui Su ◽  
Wan-Yu Wu ◽  
Tsu-Lan Wu
Keyword(s):  
Cerebrospinal Fluid ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Cloning Vector ◽  
Typhimurium Strain ◽  
Serovar Typhimurium ◽  
High Level ◽  
Lactamase Gene

ABSTRACT A Salmonella enterica serovar Typhimurium strain that harbored a plasmid carrying a TEM-1-type β-lactamase gene was isolated from the blood and cerebrospinal fluid of an infant with meningitis. This 3.2-kb plasmid was further characterized to be a nonconjugative pGEM series cloning vector containing a foreign insert. The strain was likely laboratory derived and contaminated the environment before it caused the infection.

scholarly journals A successful use of a new shuttle cloning vector pA13 for the cloning of the bacteriocins BacSJ and acidocin 8912

2010 ◽  
Vol 62 (2) ◽  
pp. 231-243 ◽  
Author(s):  
Milan Kojic ◽  
Jelena Lozo ◽  
B. Jovcic ◽  
Ivana Strahinic ◽  
D. Fira ◽  
...  
Keyword(s):  
In Silico Analysis ◽  
Functional Expression ◽  
Open Reading Frames ◽  
Cloning Vector ◽  
The Novel ◽  
Genetic Manipulations ◽  
Genes Encoding ◽  
Editorial Decision ◽  
Terminal Amino

The aim of this paper was to research the molecular cloning of genes encoding the novel bacteriocin BacSJ from Lactobacillus paracasei subsp. paracasei BGSJ2-8 by using a newly constructed shuttle cloning vector pA13. A new shuttle-cloning vector, pA13, was constructed and successfully introduced into Escherichia coli, Lactobacillus and Lactococcus strains, showing a high segregational and structural stability in all three hosts. The natural plasmid pSJ2-8 from L. paracasei subsp. paracasei BGSJ2-8 was cloned in the pA13 using BamHI, obtaining the construct pB5. Sequencing and in silico analysis of the pB5 revealed 15 open reading frames (ORF). Plasmid pSJ2-8 harbors the genes encoding the production of two bacteriocins, BacSJ and acidocin 8912. The combined N-terminal amino acid sequencing of BacSJ in combination with DNA sequencing of the bacSJ2-8 gene enabled the determination of the primary structure of a bacteriocin BacSJ. The production and functional expression of BacSJ in homologous and heterologous hosts suggest that bacSJ2-8 and bacSJ2-8i together with the genes encoding the ABC transporter and accessory protein are the minimal requirement for the production of BacSJ. Biochemical and genetic analyses showed that BacSJ belongs to the class II bacteriocins. The shuttle cloning vector pA13 could be used as a tool for genetic manipulations in lactobacilli and lactococci. <br><br><b><font color="red">withdrawn; due to a printing error. Link to the Editorial Decision <u><a href="http://dx.doi.org/10.2298/ABS1004251U">10.2298/ABS1004251U</a></u></font></b><br>

A common multiple cloning site in a set of vectors for expression of eukaryotic genes in mammalian, insect and bacterial cells

Gene ◽  
1994 ◽  
Vol 138 (1-2) ◽  
pp. 115-118 ◽  
Author(s):  
Niels Pallisgaard ◽  
Finn Skou Pedersen ◽  
Svend Birkelund ◽  
Poul Jørgensen
Keyword(s):  
Multiple Cloning Site ◽  
Bacterial Cells ◽  
Common Multiple ◽  
Eukaryotic Genes
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