scholarly journals COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast

2019 ◽  
Author(s):  
Youlian Goulev ◽  
Audrey Matifas ◽  
Vincent Heyer ◽  
Bernardo Reina-San-Martin ◽  
Gilles Charvin
Keyword(s):  
Expression Vector ◽  
Experimental Approach ◽  
Target Location ◽  
Single Copy ◽  
Expression Vectors ◽  
Functional Modules ◽  
Golden Gate ◽  
Genetic Studies ◽  
Promoter Sequences ◽  
Expression Plasmids

AbstractA large number of genetic studies in yeast rely on the use of expression vectors. To facilitate the experimental approach of these studies, several collections of expression vectors have been generated (YXplac, pRS series, etc.). Subsequently, these collections have been expanded by adding more diversity to many of the plasmid features, including new selection markers and new promoter sequences. However, the ever growing number of plasmid feature makes it unrealistic for research labs to maintain an up-to-date collection of plasmids.. Here, we developed the COSPLAY toolbox: a Golden Gate approach based on the scheme of a simple modular plasmid that recapitulates and completes all the properties of the pRS plasmids. The COSPLAY toolbox contains a basal collection of individual functional modules. Moreover we standardized a simple and rapid, software-assisted protocol which facilitates the addition of new personalized modules. Finally, our toolbox includes the possibility to select a genomic target location and to perform a single copy integration of the expression vector.

scholarly journals pET expression vector customized for efficient seamless cloning

BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 384-387
Author(s):  
Dragana Dobrijevic ◽  
Lily A Nematollahi ◽  
Helen C Hailes ◽  
John M Ward
Keyword(s):  
Expression Vector ◽  
Gene Replacement ◽  
Expression Vectors ◽  
Gram Negative Bacteria ◽  
Golden Gate ◽  
Gram Negative ◽  
Large Numbers ◽  
Parallel Cloning ◽  
Natural Enzyme

Here we present a modification of the widely used pET29 expression vector for use in rapid and straightforward parallel cloning via a gene replacement and Golden Gate strategy. The modification can be applied to other expression vectors for Gram-negative bacteria. We have used the modified vectors to clone large numbers of bacterial natural enzyme variants from genomic and metagenomic sources for applications in biocatalysis.

scholarly journals Whole-genome assembly of Ganoderma leucocontextum (Ganodermataceae, Fungi) discovered from the Tibetan Plateau of China

2021 ◽  
Author(s):  
Yuanchao Liu ◽  
Longhua Huang ◽  
Huiping Hu ◽  
Manjun Cai ◽  
Xiaowei Liang ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Southwest China ◽  
Reference Genome ◽  
Biological Activities ◽  
Single Copy ◽  
The Tibetan Plateau ◽  
Whole Genome ◽  
High Quality ◽  
Pharmacological Activities ◽  
Genetic Studies

Abstract Ganoderma leucocontextum, a newly discovered species of Ganodermataceae in China, has diverse pharmacological activities. G. leucocontextum was widely cultivated in southwest China, but the systematic genetic study has been impeded by the lack of a reference genome. Herein, we present the first whole-genome assembly of G. leucocontextum based on the Illumina and Nanopore platform from high-quality DNA extracted from a monokaryon strain (DH-8). The generated genome was 50.05 Mb in size with a N50 scaffold size of 3.06 Mb, 78,206 coding sequences and 13,390 putative genes. Genome completeness was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) tool, which identified 96.55% of the 280 Fungi BUSCO genes. Furthermore, differences in functional genes of secondary metabolites (terpenoids) were analyzed between G. leucocontextum and G. lucidum. G. leucocontextum has more genes related to terpenoids synthesis compared to G. lucidum, which may be one of the reasons why they exhibit different biological activities. This is the first genome assembly and annotation for G. leucocontextum, which would enrich the toolbox for biological and genetic studies in G. leucocontextum.

scholarly journals Use of Intron-Disrupted Polyadenylation Sites To Enhance Expression and Safety of Retroviral Vectors

2001 ◽  
Vol 75 (1) ◽  
pp. 199-204 ◽  
Author(s):  
Said I. Ismail ◽  
Jonathan B. Rohll ◽  
Susan M. Kingsman ◽  
Alan J. Kingsman ◽  
Mark Uden
Keyword(s):  
Site Selection ◽  
Expression Vector ◽  
Retroviral Vectors ◽  
Expression Vectors ◽  
Enhanced Expression ◽  
Producer Cell ◽  
Polyadenylation Sites ◽  
Internal Signal ◽  
Mrna Polyadenylation ◽  
Polyadenylation Signals

ABSTRACT Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256–5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy.

scholarly journals Construction and Characterization of a Gradually Inducible Expression Vector for Halobacterium salinarum, Based on thekdpPromoter

2012 ◽  
Vol 78 (7) ◽  
pp. 2100-2105 ◽  
Author(s):  
Dorthe Kixmüller ◽  
Jörg-Christian Greie
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Expression System ◽  
Expression Patterns ◽  
Inducible Expression ◽  
Halobacterium Salinarum ◽  
Expression Vectors ◽  
Content Type ◽  
Inducible Expression System

ABSTRACTGradually inducible expression vectors which are governed by variations of growth conditions are powerful tools for gene expression of conditionally lethal mutants. Furthermore, controlled expression allows monitoring of overproduction of proteins at various stages in their expressing hosts. ForHalobacterium salinarum, which is often used as a paradigm for halophilic archaea, such an inducible expression system is not available to date. Here we show that thekdppromoter (Pkdp), which facilitates gene expression upon K+limitation, can be used to establish such a system for molecular applications. Pkdpfeatures a rather high expression rate, with an approximately 50-fold increase that can be easily varied by K+concentrations in the growth medium. Besides the construction of an expression vector, our work describes the characterization of expression patterns and, thus, offers a gradually inducible expression system to the scientific community.

Development of acetosyringone-inducible Gateway® and Golden Gate expression vectors for heterologous gene expression in Agrobacterium tumefaciens

2020 ◽  
Vol 56 (5) ◽  
pp. 578-587
Author(s):  
Wai Keat Toh ◽  
Eliza Po-Iian Loo ◽  
Chong Siang Tee ◽  
Pek Chin Loh ◽  
Hann Ling Wong
Keyword(s):  
Gene Expression ◽  
Agrobacterium Tumefaciens ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
Expression Vectors ◽  
Golden Gate

scholarly journals Human Papillomavirus Type 31 Replication Modes during the Early Phases of the Viral Life Cycle Depend on Transcriptional and Posttranscriptional Regulation of E1 and E2 Expression

2002 ◽  
Vol 76 (5) ◽  
pp. 2263-2273 ◽  
Author(s):  
Walter G. Hubert ◽  
Laimonis A. Laimins
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Expression Vector ◽  
Viral Life Cycle ◽  
Expression Vectors ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Transient Assays ◽  
Splicing Mutations ◽  
Efficient Expression

ABSTRACT The E1 and E2 proteins are both required for papillomavirus DNA replication, and replication efficiency is controlled by the abundance of these factors. In human papillomaviruses (HPVs), the regulation of E1 and E2 expression and its effect on viral replication are not well understood. In particular, it is not known if E1 and E2 modulate their own expression and how posttranscriptional mechanisms may affect the levels of the replication proteins. Previous studies have implicated splicing within the E6 open reading frame (ORF) as being important for modulating replication of HPV type 31 (HPV31) through altered expression of E1 and E2. To analyze the function of the E6 intron in viral replication more specifically, we examined the effects of E6 splicing mutations in the context of entire viral genomes in transient assays. HPV31 genomes which had mutations in the splice donor site (E6SD) or the splice acceptor site (E6SA), a deletion of the intron (E6ID), or substituted heterologous intron sequences (E6IS) were constructed. Compared to wild-type (wt) HPV31, pHPV31-E6SD, -E6SA, and -E6IS replicated inefficiently while pHPV31-E6ID replicated at an intermediate level. Cotransfection of the E6 mutant genomes with an E1 expression vector strongly activated their replication levels, indicating that efficient expression of E1 requires E6 internal splicing. In contrast, replication was activated only moderately with an E2 expression vector. Replacing the wt E6 intron in HPV31 with a heterologous intron from simian virus 40 (E6SR2) resulted in replication levels similar to that of the wt in the absence of expression vectors, suggesting that mRNA splicing upstream of the E1 ORF is important for high-level replication. To examine the effects of E6 intron splicing on E1 and E2 expression directly, we constructed reporter DNAs in which the luciferase coding sequences were fused in frame to the E1 (E1Luc) or E2 (E2Luc) gene. Reporter activities were then analyzed in transient assays with cotransfected E1 or E2 expression vectors. Both reporters were moderately activated by E1 in a dose-dependent manner. In addition, E1Luc was activated by low doses of E2 but was repressed at high doses. In contrast, E2 had little effect on E2Luc activity. These data indicate that E1 expression and that of E2 are interdependent and regulated differentially. When the E6 splicing mutations were analyzed in both reporter backgrounds, only E1Luc activities correlated with splicing competence in the E6 ORF. These findings support the hypothesis that the E6 intron primarily regulates expression of E1. Finally, in long-term replication assays, none of the E6 mutant genomes could be stably maintained. However, cotransfection of the E6 splicing mutant genomes with pHPV31-E7NS, which contains a nonsense mutation in the E7 coding sequence, restored stable replication of some mutants. Our observations indicate that E1 expression and that of E2 are differentially regulated at multiple levels and that efficient expression of E1 is required for transient and stable viral replication. These regulatory mechanisms likely act to control HPV copy number during the various phases of the viral life cycle.

Development of single-copy RFLP markers for population genetic studies of Phialocephala fortinii and closely related taxa

2003 ◽  
Vol 107 (11) ◽  
pp. 1332-1341 ◽  
Author(s):  
Christoph R. GrÜNig ◽  
Celeste C. Linde ◽  
Thomas N. Sieber ◽  
Scott O. Rogers
Keyword(s):  
Population Genetic ◽  
Single Copy ◽  
Rflp Markers ◽  
Genetic Studies ◽  
Phialocephala Fortinii

scholarly journals Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytoadherence.

1988 ◽  
Vol 167 (2) ◽  
pp. 718-723 ◽  
Author(s):  
S F Dallo ◽  
C J Su ◽  
J R Horton ◽  
J B Baseman
Keyword(s):  
Amino Acids ◽  
Mycoplasma Pneumoniae ◽  
Synthetic Peptide ◽  
Fusion Proteins ◽  
Genomic Dna ◽  
Expression Vector ◽  
Genomic Library ◽  
Vaccine Candidate ◽  
Single Copy

A genomic library of Mycoplasma pneumoniae was constructed by cloning sheared genomic DNA into the expression vector lambda gt11. Recombinant clones were screened using anti-M. pneumoniae mAbs reactive with adhesin P1 epitopes that mediate cytadherence. 10 clones with different size inserts were isolated. These clones possessed P1 sequences localized to the COOH terminus of the P1 gene. All clones produced fusion proteins that reacted with acute and convalescent sera of patients infected with M. pneumoniae. Interestingly, one clone, P1-7, contained an epitope that was confined to a region of 13 amino acids present in the M. pneumoniae genome as a single copy. The identification of this cytadherence-related epitope permits the production of a synthetic peptide that can be used as a rational vaccine candidate and serodiagnostic probe.

scholarly journals Complete Chloroplast Genome Sequence of Erigeron breviscapus and Characterization of Chloroplast Regulatory Elements

2021 ◽  
Vol 12 ◽  
Author(s):  
Yifan Yu ◽  
Zhen Ouyang ◽  
Juan Guo ◽  
Wen Zeng ◽  
Yujun Zhao ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Single Copy ◽  
Regulatory Elements ◽  
Rrna Genes ◽  
Expression Vectors ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Flanking Sequences ◽  
Erigeron Breviscapus ◽  
Cp Genome

Erigeron breviscapus is a famous medicinal plant. However, the limited chloroplast genome information of E. breviscapus, especially for the chloroplast DNA sequence resources, has hindered the study of E. breviscapus chloroplast genome transformation. Here, the complete chloroplast (cp) genome of E. breviscapus was reported. This genome was 152,164bp in length, included 37.2% GC content and was structurally arranged into two 24,699bp inverted repeats (IRs) and two single-copy areas. The sizes of the large single-copy region and the small single-copy region were 84,657 and 18,109bp, respectively. The E. breviscapus cp genome consisted of 127 coding genes, including 83 protein coding genes, 36 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. For those genes, 95 genes were single copy genes and 16 genes were duplicated in two inverted regions with seven tRNAs, four rRNAs, and five protein coding genes. Then, genomic DNA of E. breviscapus was used as a template, and the endogenous 5' and 3' flanking sequences of the trnI gene and trnA gene were selected as homologous recombinant fragments in vector construction and cloned through PCR. The endogenous 5' flanking sequences of the psbA gene and rrn16S gene, the endogenous 3' flanking sequences of the psbA gene, rbcL gene, and rps16 gene and one sequence element from the psbN-psbH chloroplast operon were cloned, and certain chloroplast regulatory elements were identified. Two homologous recombination fragments and all of these elements were constructed into the cloning vector pBluescript SK (+) to yield a series of chloroplast expression vectors, which harbored the reporter gene EGFP and the selectable marker aadA gene. After identification, the chloroplast expression vectors were transformed into Escherichia coli and the function of predicted regulatory elements was confirmed by a spectinomycin resistance test and fluorescence intensity measurement. The results indicated that aadA gene and EGFP gene were efficiently expressed under the regulation of predicted regulatory elements and the chloroplast expression vector had been successfully constructed, thereby providing a solid foundation for establishing subsequent E. breviscapus chloroplast transformation system and genetic improvement of E. breviscapus.

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