scholarly journals EXPRESSION AND PURIFICATION OF THE IPT GENE

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 580g-581
Author(s):  
Sandra L. Barbour ◽  
D.A. Schaff ◽  
A.C. Smigocki ◽  
J.J. Frett
Keyword(s):  
Agrobacterium Tumefaciens ◽  
New England ◽  
Cloning And Expression ◽  
Isopentenyl Transferase ◽  
Cytokinin Biosynthesis ◽  
E Coli ◽  
Shoot Production ◽  
Cytokinin Synthesis ◽  
Polymerase Chain

The ipt gene of Agrobacterium tumefaciens T-DNA encodes for isopentenyl transferase, which is an enzyme active in cytokinin biosynthesis. While it is known that cytokinins are associated with in vitro promotion of cell division and stimulation of shoot production, little is known about their mode of action. As the first step in localizing cytokinin synthesis, we present a cloning and expression strategy for the ipt gene. The source of the ipt gene was Agrobacterium tumefaciens octopine Ti plasmid 15955. The ipt gene was amplified by the polymerase chain reaction (PCR) and cloned into pMal-c2 (New England Biolabs, Beverly, MA). This construct was transformed into E. coli and the ipt gene was expressed as a fusion protein. The protein was purified by affinity chromatography to serve as an antigen for polyclonal antibody production. These antibodies will be used to localize isopentenyl transferase in plant tissue.

scholarly journals CLONING AND EXPRESSION OF THE IPT GENE

HortScience ◽  
1992 ◽  
Vol 27 (11) ◽  
pp. 1160e-1160
Author(s):  
Sandra L. Barbour ◽  
D.A. Schaff ◽  
J.J. Frett
Keyword(s):  
New England ◽  
Cloning And Expression ◽  
Isopentenyl Transferase ◽  
Shoot Initiation ◽  
Purification System ◽  
England Biolabs ◽  
Cytokinin Synthesis ◽  
Polymerase Chain ◽  
Future Work

Cytokinins are involved in in vitro shoot initiation, although little is known about their mode of action. As the first step in localizing cytokinin synthesis, we present a cloning and expression strategy for the isopentenyl transferase (ipt) gene. The source of the Agrobacterium tumefaciens ipt gene was a 7.2 kb Eco RI fragment isolated from pBREF7 (Dr. Ann Smigocki, USDA, Beltsville, MD). The ipt gene was amplified by polymerase chain reaction (PCR) and cloned into pMal-cRI (New England Biolabs, Beverly, MA). Using the pMAL-cRI expression and purification system, the isopentenyl transferase protein will be purified for antibody production. These antibodies will be used in future work to localize the isopentenyl transferase enzyme.

scholarly journals PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST IPTASE

HortScience ◽  
1993 ◽  
Vol 28 (4) ◽  
pp. 257F-257
Author(s):  
Sandra L. Barbour ◽  
John J. Frett
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fusion Protein ◽  
New England ◽  
Polyclonal Antibodies ◽  
Isopentenyl Transferase ◽  
Cytokinin Biosynthesis ◽  
Western Blots ◽  
Sds Page ◽  
England Biolabs ◽  
Secondary Antibodies

Isopentenyl transferase, encoded by the ipt gene of Agrobacterium tumefaciens T-DNA, is an enzyme active in cytokinin biosynthesis. The ipt gene was cloned into the pMAL-c2 vector (New England Biolabs, Beverly, MA) and expressed as a fusion protein. The production of this fusion protein was induced by a 2 hour exposure to IPTG. The fusion protein was then purified by a mini-aggregate procedure and visualized by SDS-PAGE. To verify that the correct protein was purified, antibodies specific to the conserved region of the fusion protein were used to probe a western blot. Secondary antibodies were biotinylated. Rabbits were immunized to raise polyclonal antibodies against iptase. Using a slot format blotting apparatus, serum was titered. These antibodies will be used to probe western blots from transgenic plants transformed with various ipt constructs.

scholarly journals Different Regulation and Roles of Lactonases AiiB and AttM in Agrobacterium tumefaciens C58

2009 ◽  
Vol 22 (5) ◽  
pp. 529-537 ◽  
Author(s):  
Elise Haudecoeur ◽  
Mélanie Tannières ◽  
Amélie Cirou ◽  
Aurélie Raffoux ◽  
Yves Dessaux ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Selective Advantage ◽  
In Planta ◽  
Host Interaction ◽  
Plant Tumors ◽  
Plant Signals ◽  
Polymerase Chain ◽  
Agrobacterium Tumefaciens C58 ◽  
Regulated Functions

The phytopathogen Agrobacterium tumefaciens C58 expresses two lactonases, AttM and AiiB. We showed that expression of the aiiB gene was controlled by agrocinopines A and B and required the agrocinopine-ABC transporter Acc, but was not affected by the level of quorum-sensing (QS) signal 3-oxo-octanoylhomoserine lactone (OC8-HSL). In the presence of agrocinopines, a constructed aiiB mutant accumulated OC8-HSL at a level 10-fold higher than that of the wild-type strain, and showed an exacerbated expression of a key QS-regulated function, conjugation of Ti plasmid (in vitro and in planta), as well as an increase of the number of emerging tumors on the host plant. The expression and acyl-HSL-degrading activity of AttM were evident in the presence of wounded tissues; however, in unwounded plant tumors, the QS-regulated functions were weakly affected in an attM mutant. By contrast, we observed that attM conferred a selective advantage in the course of colonization of plant tumors. Finally, polymerase chain reaction survey of genes attM and aiiB showed that they were not strictly conserved in the genus Agrobacterium. This work proved that the lactonases AttM and AiiB are regulated by different plant signals and are implicated in different functions in the course of the A. tumefaciens C58–host interaction.

scholarly journals Transformation of Nicotiana alata Link and Otto. with an Autoregulatory Senescence-inhibitor Gene Construct

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 617c-617
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Leaf Explants ◽  
Shoot Proliferation ◽  
Nicotiana Alata ◽  
Ms Medium ◽  
Gene Encoding ◽  
Half Strength ◽  
Cytokinin Synthesis ◽  
Senesced Leaves

Leaf explants of Nicotiana alata Link and Otto. were surface disinfested and cultured on Murashige and Skoog (MS) medium containing 2.66 μm N6-benzyladenine (BA) to promote shoot proliferation. After 5 weeks, proliferated shoots were removed and remaining callus saved. Callus was inoculated with Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase which catalyzes cytokinin synthesis. Following inoculation, the callus was cocultivated for 6 days on BA medium. Selection for transgenics was done on BA medium plus 100 mg Kanamycin and 400 mg Ticarcillin (antibiotics) per liter. Proliferating shoots were rooted on MS medium containing antibiotics. Rooted cuttings were transplanted to soil, acclimated and flowered in the greenhouse. Transgenics were outcrossed to a commercial N. alata hybrid. Seed was germinated in vitro on half-strength MS medium plus antibiotics. Segregation of transgenics to nontransgenics was 1:1. Evaluation of leaf senescence on 5-month-old plants showed 2 to 14 times fewer senesced leaves on the transgenic than the nontransgenic plants.

scholarly journals Identification of a Two-Partner Secretion Locus of Enterotoxigenic Escherichia coli

2006 ◽  
Vol 74 (4) ◽  
pp. 2245-2258 ◽  
Author(s):  
James M. Fleckenstein ◽  
Koushik Roy ◽  
Julia F. Fischer ◽  
Michael Burkitt
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Epithelial Cells ◽  
High Molecular Weight ◽  
Cloning And Expression ◽  
Peptide Sequence ◽  
Enterotoxigenic Escherichia Coli ◽  
Virulence Plasmid ◽  
E Coli

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) remains a formidable cause of diarrheal illness worldwide. At present, there is no vaccine that provides broad-based protection against ETEC. A ′phoA-based self-cloning mutagenesis system, TnphoA.ts, employed to identify novel ETEC surface antigens, led to identification of an ETEC two-partner secretion locus (etpBAC) on the pCS1 virulence plasmid of prototype strain H10407. Cloning and expression of etpBAC in recombinant E. coli LMG194(pJY019) resulted in secretion of a high-molecular-weight (HMW) glycosylated exoprotein. This glycoprotein, EtpA, exhibits linear peptide sequence and predicted structural homologies with known HMW adhesins produced by other two-partner secretion loci. Antibodies directed against recombinant EtpA (anti-rEtpA.6H) recognized an HMW protein in culture supernatants of ETEC strains H10407 and LMG194(pJY019) but not in culture supernatant of strain H10407-P, which lacks the 92-kb pCS1 plasmid, or an isogenic etpA mutant. etpA mutants were deficient in adherence to intestinal epithelial cells in vitro, and anti-rEtpA.6H antibodies inhibited association of H10407 with target epithelial cells. Cloning and expression of etpB in recombinant E. coli were sufficient to confer adherence. Screening of multiple ETEC isolates for the etpBAC locus by colony hybridization and by EtpA immunoblotting suggested that EtpA is one of the most common antigens secreted by these pathogens. Together, these results indicate that the newly identified ETEC two-partner secretion locus directs the secretion of a high-molecular-weight glycosylated protein, EtpA, that in concert with the putative EtpB transporter participates in adherence of H10407 to epithelial cells, thereby expanding the repertoire of potential ETEC virulence proteins and vaccine candidates.

Preparation and Standardization of Escherichia coli Nosodes Sourced from Select E. coli Strains

Homeopathy ◽  
2020 ◽  
Vol 109 (04) ◽  
pp. 207-212
Author(s):  
Renuka Munshi ◽  
Gitanjali Talele ◽  
Rajesh Shah
Keyword(s):  
Escherichia Coli ◽  
Preparation Method ◽  
Cell Extract ◽  
Molecular Testing ◽  
Chain Reaction ◽  
E Coli ◽  
Entire Cell ◽  
Polymerase Chain ◽  
Different Strains

Abstract Background The nosodes are well-known preparations in homeopathy that are sourced from organisms and diseased materials. More than 40 known nosodes have been used in homeopathic practice for over a century. Having identified the need for scientifically developed new nosodes sourced from organisms that are currently prevalent, the preparation of Escherichia coli nosodes from different strains of the bacterium is presented in this article. Materials and Methods Escherichia coli strains (E. coli ATCC 11775E, ATCC 25922, and ATCC 8739) were identified, cultured, and tested for purity, and 20 billion cells were processed following the nosode preparation method given in the Homoeopathic Pharmacopoeia of India, group N1. Serial dilution and potentization for liquid potency were done up to 30c potency. Nosodes were prepared by two methods: from cell-free extract (endotoxin) and from entire-cell extract. Result Six nosodes were developed in total. Three univalent nosodes were prepared using individual endotoxins, one from each of the three E. coli strains; those three univalent nosodes were also combined as “Trivalent nosode-I”. “Trivalent nosode-II” was prepared by mixing entire cells of the three E. coli strains. A mix of both Trivalent nosode-I and Trivalent nosode-II was labeled “EC-Polynosode”. The safety profile of the potentized nosodes was documented by the non-detectability of traces of source material (absence of contamination, live organisms, or DNA material) through a culture test, sterility test, and molecular testing (polymerase chain reaction). Conclusion Different variants of E. coli nosodes were systematically and scientifically prepared and standardized using the cultures. Homeopathic pathogenetic trials, in-vitro efficacy studies, and clinical evaluation of E. coli nosodes (single, trivalent, or polyvalent nosodes) will be required in future.

Cloning and expression ofSpodoptera lituranucleopolyhedrovirusgp41gene inEscherichia coliand preparation of antibody

2005 ◽  
Vol 2 (2) ◽  
pp. 113-117
Author(s):  
Pan Li-Jing ◽  
Li Zhao-Fei ◽  
Yin Chong ◽  
Lv Lei ◽  
Pang Yi
Keyword(s):  
Fusion Protein ◽  
Prokaryotic Expression ◽  
Recombinant Plasmid ◽  
Nitrilotriacetic Acid ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Pcr Product ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain

AbstractGP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) ofSpodoptera lituranucleopolyhedrovirus (SpltMNPV)gp41gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). Thegp41gene was recombinedin vitrowith prokaryotic expression vector pQE30 and transformed intoEscherichia coliM15 [pREP4]. The M15 [pREP4] strain, containinggp41recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

scholarly journals The prevalence and mechanism of triclosan resistance in Escherichia coli isolated from urine samples in Wenzhou, China

2020 ◽  
Author(s):  
Weiliang Zeng ◽  
Wenya Xu ◽  
Ye Xu ◽  
Wenli Liao ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Urine Samples ◽  
Cross Resistance ◽  
Chain Reaction ◽  
E Coli ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Triclosan Resistance ◽  
Encoding Genes

Abstract Background: The widespread application of triclosan contributes to its residual deposition in urine, which provides an environment of long-term exposure to triclosan for the intestinal Escherichia coli. We determined the triclosan and antibiotic resistance characteristics of E. coli strains isolated from urine samples and further investigated the resistance mechanism and molecular epidemic characteristics of triclosan-resistant E. coli isolates. Methods: A total of 200 non-repetitive E. coli strains were isolated from urine samples and then identified. The minimum inhibitory concentrations (MICs) of triclosan and antibiotics, fabI mutation, efflux pump activity, the expression of 14 efflux pump encoding genes, and epidemiological characteristics were determined by the agar dilution method, polymerase chain reaction (PCR), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibition test, quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing (MLST), and pulse-field gel electrophoresis (PFGE) for all triclosan-resistant isolates. Furthermore, we also investigated the effect of triclosan exposure in vitro on antibiotic susceptibility and the efflux pump encoding gene expressions of triclosan-susceptible strains via serial passage experiments. Results: Of the 200 E. coli isolates, 2.5% (n = 5) were found to be resistant to triclosan, and multidrug resistance (MDR) and cross-resistance phenotypes were noted for these triclosan-resistant strains. The triclosan-sensitive strains also exhibited MDR phenotypes, probably because of the high resistance rate to AMP, CIP, LVX, and GEN. Gly79Ala and Ala69Thr amino acid changes were observed in the triclosan-resistant strains, but these changes may not mediate resistance of E. coli to triclosan, because mutations of these two amino acids has also been detected in triclosan-susceptible strains. Moreover, except for DC8603, all other strains enhanced the efflux pumps activity. As compared with ATCC 25922, except for fabI, increased expressions were noted for all efflux pump encoding genes such as ydcV, ydcU, ydcS, ydcT, cysP, yihV, acrB, acrD, and mdfA among the studied strains with varying PFGE patterns and STs types. Unexpectedly, 5 susceptible E. coli isolates showed rapidly increasing triclosan resistance after exposure to triclosan in vitro for only 12 days, while MDR or cross-resistance phenotypes and the overexpression of efflux pump genes were recorded among these triclosan-induced resistant isolates. Conclusions: This is the first study to report that short-term triclosan exposure in vitro increases triclosan resistance in susceptible E. coli isolates. After acquiring resistance, these strains may present MDR or cross-resistance phenotypes. Moreover, triclosan resistance mainly involves the overexpression of fabI and efflux pumps in E. coli isolates.

scholarly journals Growth and In Vitro Propagation of Peach Plants Transformed with the Shooty Mutant Strain of Agrobacterium tumefaciens

HortScience ◽  
1998 ◽  
Vol 33 (5) ◽  
pp. 897-899 ◽  
Author(s):  
F.A. Hammerschlag ◽  
A.C. Smigocki
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Mutant Strain ◽  
In Vitro Propagation ◽  
Prunus Persica ◽  
Shoot Formation ◽  
Fresh Mass ◽  
Axillary Shoot ◽  
Shoot Cultures ◽  
Cytokinin Biosynthesis

Peach [Prunus persica (L.) Batsch] plants #94-1, #99-1, and #40-1, carrying a cytokinin biosynthesis (ipt) gene following transformation with the shooty mutant strain of Agrobacterium tumefaciens, were evaluated for altered growth habit and axillary shoot formation, both in vitro and in the greenhouse. After 9 weeks of in vitro propagation on four different levels of 6-benzyladenine (BA), only transformant #99-1 exhibited significantly greater axillary shoot formation (on 10 μm BA), and significantly greater fresh mass (on 3,10, and 30 μm BA) than the control #RG-3. Tolerance to a supra-optimal (30 μm) concentration of BA was indicated by fresh mass increases for #99-1 shoot cultures. Delayed senescence on 0 μm BA was exhibited by 87% of the transformants, but by only 12% of the control plants. Greenhouse-grown #99-1 and #40-1 were significantly shorter than #RG-3 plants at 6 weeks and at 1 year, but only #40-1 exhibited significantly greater branching than the controls. Chemical names used: 6-benzyladenine (BA); isopentenyl transferase (ipt).

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