scholarly journals Efficient Method for Genomic DNA Mutagenesis in E. coli

2020 ◽  
Author(s):  
Dave Palis ◽  
Frank Huang
Keyword(s):  
Efficient Method ◽  
Genomic Dna ◽  
E Coli ◽  
Citation Statistics ◽  
Dna Mutagenesis

This paper has been withdrawn by bioRxiv because its content, including the author names, was fabricated and fraudulently submitted in what may have been an attempt to game citation statistics or other metrics

Cloning and expression of Xylella fastidiosa antigens in Escherichia coli and Erwinia stewartii

1989 ◽  
Vol 35 (4) ◽  
pp. 487-491 ◽  
Author(s):  
Paul H. Goodwin
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Genomic Dna ◽  
Xylella Fastidiosa ◽  
Gene Bank ◽  
Cloning And Expression ◽  
Western Blots ◽  
E Coli ◽  
Cosmid Vector ◽  
Erwinia Stewartii

Xylella fastidiosa DNA, partially digested with Sau3A, was ligated into the cosmid vector, pUCD615. Approximately 4500 ampicillin-resistant Escherichia coli colonies were obtained. The frequency of complementation of leucine auxotrophy in transfected E. coli indicated that the cosmid gene bank was representative of X. fastidiosa genomic DNA. Colonies were lysed directly onto nitrocellulose membranes using a thermo-inducible λ lysogen and screened for expression of X. fastidiosa antigens. Approximately 16.5% of a random sample of clones were found to express X. fastidiosa antigens as determined by Western blots. These proteins comigrated with proteins of X. fastidiosa and ranged in molecular weight from 10 000 to 160 000. Conjugation of several of the plasmids into Erwinia stewartii resulted in expression of the similar molecular weight cloned proteins with similar levels of expression as in E. coli.Key words: Xylella fastidiosa, Pierce's disease, immunological clone screening, thermo-inducible lysogeny.

scholarly journals Structure of recombinants from conjugational crosses between Escherichia coli donor and mismatch-repair deficient Salmonella typhimurium recipients.

Genetics ◽  
1994 ◽  
Vol 136 (1) ◽  
pp. 17-26
Author(s):  
I Matic ◽  
M Radman ◽  
C Rayssiguier
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Mismatch Repair ◽  
Genomic Dna ◽  
Sequence Divergence ◽  
Physical Analysis ◽  
Repair System ◽  
Donor Genome ◽  
E Coli ◽  
Recombination Pathway

Abstract To get more insight into the control of homologous recombination between diverged DNA by the Mut proteins of the long-patch mismatch repair system, we have studied interspecies Escherichia coli/Salmonella typhimurium recombination. Knowing that the same recombination pathway (RecABCD) is responsible for intraspecies and interspecies recombination, we have now studied the structure (replacement vs. addition-type or other rearrangement-type recombinants) of 81 interspecies recombinants obtained in conjugational crosses between E. coli donor and mutL, mutS, mutH, mutU or mut+ S. typhimurium recipients. Taking advantage of high interspecies sequence divergence, a physical analysis was performed on one third of the E. coli Hfr genome, which was expected to be transferred to S. typhimurium F- recipients during 40 min before interruption of the mating. Probes specific for each species were hybridized on dot blots of genomic DNA, or on colonies, and the composition of the rrn operons was determined from purified genomic DNA. With very few exceptions, the structure of these interspecies recombinants corresponds to replacements of one continuous block of the recipient genome by the corresponding region of the donor genome.

scholarly journals Survival of the weakest in non-transitive asymmetric interactions among strains of E. coli

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Michael J. Liao ◽  
Arianna Miano ◽  
Chloe B. Nguyen ◽  
Lin Chao ◽  
Jeff Hasty
Keyword(s):  
Microbial Community ◽  
Cell Membrane ◽  
Genomic Dna ◽  
Dominant Species ◽  
Protein Production ◽  
Competitive Dynamics ◽  
Hierarchical Organization ◽  
Ecological Interactions ◽  
E Coli ◽  
Asymmetric Interactions

AbstractHierarchical organization in ecology, whereby interactions are nested in a manner that leads to a dominant species, naturally result in the exclusion of all but the dominant competitor. Alternatively, non-hierarchical competitive dynamics, such as cyclical interactions, can sustain biodiversity. Here, we designed a simple microbial community with three strains of E. coli that cyclically interact through (i) the inhibition of protein production, (ii) the digestion of genomic DNA, and (iii) the disruption of the cell membrane. We find that intrinsic differences in these three major mechanisms of bacterial warfare lead to an unbalanced community that is dominated by the weakest strain. We also use a computational model to describe how the relative toxin strengths, initial fractional occupancies, and spatial patterns affect the maintenance of biodiversity. The engineering of active warfare between microbial species establishes a framework for exploration of the underlying principles that drive complex ecological interactions.

An efficient method for precise gene substitution in the AcMNPV genome by homologous recombination in E. coli

2003 ◽  
Vol 113 (2) ◽  
pp. 95-101 ◽  
Author(s):  
Wuwei Wu ◽  
Jinwen Wang ◽  
Riqiang Deng ◽  
Xunzhang Wang ◽  
XiongLei He ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Efficient Method ◽  
Gene Substitution ◽  
E Coli

scholarly journals A miniaturized biotyping system for strain discrimination inEscherichia coli

1993 ◽  
Vol 111 (1) ◽  
pp. 81-88
Author(s):  
P. B. Crichton ◽  
J. M. J. Logan ◽  
D. C. Old
Keyword(s):  
Escherichia Coli ◽  
Efficient Method ◽  
High Index ◽  
Inescherichia Coli ◽  
E Coli

SummaryA two-tier miniaturized scheme of eight tests was devised for biotyping strains ofEscherichia coliin microwell plates. Primary biotypes were defined by positive and negative reactions in tests for fermentation of raffinose, sorbose, dulcitol and 2-deoxy-D-ribose and for decarboxylation of ornithine when read after specified periods of incubation; subtypes were identified within primary biotypes according to results in secondary tests for rhamnose fermentation, lysine decarboxylation and motility. The method gave reproducible results on different occasions of testing.Among 100E. colistrains from various sources, 26 of the 32 possible primary biotypes and 56 full biotypes, as defined by results in both primary and secondary tests, were identified, thus demonstrating a high index of strain discrimination (D = 0·98).The scheme is recommended as a simple, reliable, inexpensive and efficient method of differentiating strains ofE. coli.

scholarly journals Integration of a Gold-Specific Whole E. coli Cell Sensing and Adsorption Based on BioBrick

2018 ◽  
Vol 19 (12) ◽  
pp. 3741
Author(s):  
Li Yan ◽  
Peiqing Sun ◽  
Yun Xu ◽  
Shanbo Zhang ◽  
Wei Wei ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Efficient Method ◽  
Visual Detection ◽  
Specific Binding ◽  
Selective Detection ◽  
Biological Detection ◽  
Integration System ◽  
Major Task ◽  
E Coli ◽  
Specific Binding Protein

Detection and recovery of heavy metals from environmental sources is a major task in environmental protection and governance. Based on previous research into cell-based visual detection and biological adsorption, we have developed a novel system combining these two functions by the BioBrick technique. The gold-specific sensory gol regulon was assembled on the gold-chaperone GolB (Gold-specific binding protein), which is responsible for selectively absorbing gold ions, and this led to an integration system with increased probe tolerance for gold. After being incorporated into E. coli, this system featured high-selective detection and recycling of gold ions among multi-metal ions from the environment. It serves as an efficient method for biological detection and recovery of various heavy metals. We have developed modular methods for cell-based detection and adsorption of heavy metals, and these offer a quick and convenient tool for development in this area.

scholarly journals Polyphosphate:AMP Phosphotransferase as a Polyphosphate-Dependent Nucleoside Monophosphate Kinase in Acinetobacter johnsonii 210A

2005 ◽  
Vol 187 (5) ◽  
pp. 1859-1865 ◽  
Author(s):  
Toshikazu Shiba ◽  
Hiromichi Itoh ◽  
Atsushi Kameda ◽  
Keiju Kobayashi ◽  
Yumi Kawazoe ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Kinase Activity ◽  
Phosphate Binding ◽  
Binding Motifs ◽  
E Coli ◽  
Acinetobacter Johnsonii ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Coupled Reactions ◽  
Coupled Reaction

ABSTRACT We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.

Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin

1997 ◽  
Vol 43 (4) ◽  
pp. 395-399 ◽  
Author(s):  
Laura Marri ◽  
Emanuela Barboni ◽  
Tiziana Irdani ◽  
Brunella Perito ◽  
Giorgio Mastromei
Keyword(s):  
Restriction Enzyme ◽  
Dna Hybridization ◽  
Genomic Dna ◽  
Restriction Pattern ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Restriction Maps ◽  
E Coli ◽  
The One ◽  
Different Sources

Streptomyces rochei A2 endoglucanase (eglS) and β-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgsl probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis of genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.Key words: Streptomyces, cellulase genes, hybridization, restriction enzyme analysis.

Construction of Prokaryotic Expression Vector Containing Flocculation Gene FLO5 and Expression of FLO5 in E. coli

2013 ◽  
Vol 310 ◽  
pp. 157-161 ◽  
Author(s):  
Jie Xue ◽  
Wen Qiang Wei ◽  
Dong Yan Zhang ◽  
Yong Li Li ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Prokaryotic Expression ◽  
Genomic Dna ◽  
Expression Vector ◽  
Gene Fragment ◽  
Form Expression ◽  
Protein Fragments ◽  
E Coli ◽  
Sds Page ◽  
Prokaryotic Expression Vector

FLO5 has been identified as a dominant flocculation gene. The goal of this study is to clone the FLO5 gene from Saccharomyces cerevisiae and express it in E. coli. In this study, the FLO5 gene amplified by PCR from S. cerevisiae was cloned into prokaryotic expression vector pET-28a to form expression vector pET28a-FLO5, finally, transferred into E.coli BL21. Methods: FLO5 gene was amplified by PCR from genomic DNA extracted from Saccharomyces cerevisiae. The amplified FLO5 gene fragment was then recombined with clone vector pMD18-T to form clone vector pMD18-T-FLO5 amplified in E.coli JM109. After confirmed with sequencing, FLO5 fragment cut out from pMD18-T-FLO5 by enzyme EcoRI and NotI was recombined into expression vector pET-28a to form vector pET28a-FLO5. Vector pET28a-FLO5 was then transferred into E. coli BL21 and protein FLO5 was expressed in E. coli BL21 by the induction with IPTG. Expressed protein fragments separated by SDS-PAGE showed a band with the size of protein FLO5 suggesting the expression of gene FLO5. with the expected This study will lay the foundation for further research in studying flocculating effect of exogenous protein expressed by genetic engineering and making new flocculating agent through recombinant engineering.

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