New yeast/E. coli/Drosophila triple shuttle vectors for efficient generation of Drosophila P element transformation constructs

Gene ◽  
2012 ◽  
Vol 511 (2) ◽  
pp. 300-305 ◽  
Author(s):  
Achim Paululat ◽  
Jürgen J. Heinisch
Keyword(s):  
P Element ◽  
Shuttle Vectors ◽  
E Coli ◽  
Efficient Generation ◽  
New Yeast ◽  
P Element Transformation

Analysis of transcriptional regulation of the s38 chorion gene of Drosophila by P element-mediated transformation

Development ◽  
1984 ◽  
Vol 83 (Supplement) ◽  
pp. 137-146
Author(s):  
Laura Kalfayan ◽  
Barbara Wakimoto ◽  
Allan Spradling
Keyword(s):  
Transcriptional Regulation ◽  
P Element ◽  
Developmental Expression ◽  
Developmental Profile ◽  
E Coli ◽  
Flanking Sequences ◽  
Dna Fragment ◽  
P Element Transformation ◽  
Lac Z

Transcriptional regulation of the s38 chorion gene was studied using P element-mediated germline transformation. A 5·27 kb DNA fragment containing the s38 gene and 5′- and 3′-flanking sequences, was tested for its ability to be transcribed with correct developmental specificity. Five single-insert transformed lines were generated by microinjection of this DNA fragment cloned into a marked P element transformation vector. In each line, the transformed gene was transcribed according to the precise developmental pattern followed by the native s38 gene. The 1·3 kb at the 5′ end of this tested fragment was fused to the E. coli lac z gene. This fragment was also capable of initiating transcription of E. coli lac z RNA with the developmental profile of the native s38 gene. In vitro deletion studies are underway to determine which sequences in the 1·3 kb fragment are necessary for regulating the developmental expression of the gene.

scholarly journals Expression of Heterologous Cellulases inThermotogasp. Strain RQ2

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Hui Xu ◽  
Dongmei Han ◽  
Zhaohui Xu
Keyword(s):  
Enzyme Activities ◽  
Bacterial Cells ◽  
Recombinant Enzymes ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Shuttle Vectors ◽  
E Coli ◽  
Chimeric Enzymes ◽  
Coding Regions ◽  
First Time ◽  
Culture Supernatants

The ability ofThermotogaspp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) fromCaldicellulosiruptor saccharolyticuswere cloned intoT.sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried byThermotoga-E. colishuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed inE. coliDH5αandT.sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. InE. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas inT.sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost inT.sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed inThermotoga, demonstrating the feasibility of using engineeredThermotogaspp. for efficient cellulose utilization.

Identification and Evaluation of Probiotic Potential in Yeast Strains Found in Kefir Drink Samples from Malaysia

2019 ◽  
Vol 15 (7) ◽  
Author(s):  
Mohd Akmal Azhar ◽  
Mimi Sakinah Abdul Munaim
Keyword(s):  
Antimicrobial Activity ◽  
Probiotic Properties ◽  
E Coli ◽  
Probiotic Potential ◽  
Rdna Sequences ◽  
Yeast Strains ◽  
16S Rdna Sequences ◽  
Human Body Temperature ◽  
Probiotic Yeast ◽  
New Yeast

AbstractKefir drink is a source of probiotic microorganism with remarkable functional and technological properties. The objective of this work is to isolate yeast strains from Malaysian kefir drink and evaluate them for probiotic potentials. In the present study, nine strains of probiotic yeast were isolated from a Malaysian kefir drink and identified according to their 16S rDNA sequences. Furthermore, their probiotic potential was evaluated. The probiotic properties were tested for aspects of antibiotic susceptibility, antimicrobial activity, and gastrointestinal condition tolerance (pH and temperature). Five isolated strains, M3, Y5, Y9, Y11 and A1, showed good tolerance towards low pH condition while three strains, A1, M1, and M3, showed antimicrobial activity against E. coli, P. aeruginosa, and Salmonella sp. Most isolates were resistant to penicillin, streptomycin, and ampicillin, and grew well at human body temperature. The result of this test indicates that the yeast strains isolated from Malaysian kefir drink have excellent potential for use as probiotics in various products. Lastly, kefir milk is one of the excellent source of probiotic yeast strains and could be used as a new yeast probiotic formulation or in food supplements.

scholarly journals pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

2001 ◽  
Vol 67 (3) ◽  
pp. 1262-1267 ◽  
Author(s):  
Shuhei Fujimoto ◽  
Yasuyoshi Ike
Keyword(s):  
Affinity Chromatography ◽  
Protein Purification ◽  
Enterococcus Faecalis ◽  
Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Chromatography Method ◽  
Shuttle Vectors ◽  
E Coli ◽  
One Step

ABSTRACT Two novel Enterococcus faecalis-Escherichia colishuttle vectors that utilize the promoter and ribosome binding site ofbacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli andE. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coliand E. faecalis at levels of 103 and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.

Characterization of plasmids from human infant Bifidobacterium strains: Sequence analysis and construction of E. coli–Bifidobacterium shuttle vectors

Plasmid ◽  
2008 ◽  
Vol 60 (2) ◽  
pp. 136-148 ◽  
Author(s):  
Andrei N. Shkoporov ◽  
Boris A. Efimov ◽  
Ekaterina V. Khokhlova ◽  
James L. Steele ◽  
Lyudmila I. Kafarskaia ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Human Infant ◽  
Shuttle Vectors ◽  
E Coli

scholarly journals Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

2004 ◽  
Vol 70 (4) ◽  
pp. 2452-2463 ◽  
Author(s):  
Asuncion Martinez ◽  
Steven J. Kolvek ◽  
Choi Lai Tiong Yip ◽  
Joern Hopke ◽  
Kara A. Brown ◽  
...  
Keyword(s):  
Natural Products ◽  
Drug Discovery ◽  
Bacterial Artificial Chromosome ◽  
Environmental Dna ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Bacterial Strains ◽  
Soil Dna ◽  
Shuttle Vectors ◽  
E Coli

ABSTRACT The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.

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