Transformation of Sinorhizobium meliloti MTCC 100 and Mesorhizobium ciceri TAL 620 by CaCl2 method

The CaCl2 method, commonly used for transformation of Escherichia coli, was modified and used to develop a simpler and easier transformation method for Rhizobia sp. Two species of Rhizobia, Sinorhizobium meliloti MTCC 100 and Mesorhizobium ciceri TAL 620, were transformed with the 13.2 kb binary vector pGA482. At an optical density of 0.4, the transformation efficiencies in Sinorhizobium meliloti MTCC 100 and Mesorhizobium ciceri TAL 620 were 104 and 103, respectively. Competent cells of Sinorhizobium meliloti MTCC 100 were prepared at different growth intervals and transformed by the same vector. A maximum transformation efficiency of 104 was achieved at an optical density of 0.5.

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A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

Bioscience Reports ◽

10.1042/bsr20130098 ◽

2013 ◽

Vol 33 (6) ◽

Cited By ~ 39

Author(s):

Weng-Tat Chan ◽

Chandra S. Verma ◽

David P. Lane ◽

Samuel Ken-En Gan

Keyword(s):

Escherichia Coli ◽

Transformation Efficiency ◽

Research Paper ◽

Factors Affecting ◽

Competent Cells ◽

Common Laboratory

This research paper aims to investigate the factors affecting the production of competent cells to produce high levels of transformation efficiency in common laboratory strains.

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Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies

Biochemical Journal ◽

10.1042/bj20060660 ◽

2006 ◽

Vol 400 (1) ◽

pp. 105-114 ◽

Cited By ~ 27

Author(s):

Eric Di Luccio ◽

Robert A. Elling ◽

David K. Wilson

Keyword(s):

Escherichia Coli ◽

Sinorhizobium Meliloti ◽

Thermotoga Maritima ◽

Xylose Reductase ◽

Sulfolobus Solfataricus ◽

Sequence Comparisons ◽

E Coli ◽

Fluorescence Measurements ◽

Dual Specificity ◽

Substrate Dependence

The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2′-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRs to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.

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One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot101212 ◽

2021 ◽

Vol 2021 (11) ◽

pp. pdb.prot101212 ◽

Cited By ~ 1

Author(s):

Michael R. Green ◽

Joseph Sambrook

Keyword(s):

Escherichia Coli ◽

Convenient Method ◽

Plasmid Dna ◽

Transformation Efficiency ◽

Bacterial Cells ◽

E Coli ◽

One Step ◽

And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

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Novel one step transformation method for Escherichia coli and Staphylococcus aureus using arginine-glucose functionalized hydroxyapatite nanoparticles

Materials Science and Engineering C ◽

10.1016/j.msec.2018.10.088 ◽

2019 ◽

Vol 96 ◽

pp. 58-65 ◽

Cited By ~ 5

Author(s):

Ketaki Deshmukh ◽

Sutapa Roy Ramanan ◽

Meenal Kowshik

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Transformation Method ◽

Hydroxyapatite Nanoparticles ◽

One Step ◽

And Staphylococcus Aureus

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Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector

Plants ◽

10.3390/plants10122636 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2636

Author(s):

Ganeshan Sivanandhan ◽

Solhee Bae ◽

Chaemin Sung ◽

Su Ryun Choi ◽

Geung-Joo Lee ◽

...

Keyword(s):

Chinese Cabbage ◽

Binary Vector ◽

Transfection Efficiency ◽

Genome Structure ◽

Transformation Method ◽

Protoplast Isolation ◽

Breeding Cycle ◽

Functional Studies ◽

Gfp Reporter Gene ◽

Protoplast Transfection

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl2, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 µg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.

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Effect of Gold Nanoparticles Size Capped with Surfactant on the Transformation of Plasmid into Escherichia coli Bacteria

Annals of Materials Science & Engineering ◽

10.26420/annmaterialsscieng.2021.1041 ◽

2021 ◽

Vol 5 (1) ◽

Author(s):

Amr D ◽

◽

Attia N ◽

Seufi A ◽

Galal A ◽

...

Keyword(s):

Escherichia Coli ◽

Gold Nanoparticles ◽

Heat Shock ◽

Transformation Efficiency ◽

Transformation Process ◽

Bacterial Cells ◽

Bacterial Transformation ◽

Foreign Genes ◽

Escherichia Coli Bacteria ◽

Convenient Technique

Bacterial transformation has great importance in molecular biology, as it is used for introduction of foreign genes into bacterial cells either chemical or physical ways. Using calcium chloride to prepare competent cells and heat shock is the most widely used method for bacterial transformation. This method is an efficient and convenient technique but it has in some extent low transformation efficiency. Here we report the use of nanoparticles that significantly improve the transformation efficiency up to 10 times higher than the standard heat shock method by the assistance of (˜ 15, 25 nm) SDS capped gold nanoparticles in the transformation process that leads to the formation of temporary nano-channels across the bacterial cell wall to deliver plasmids into cells. Transformation of bacteria with plasmid was examined using Β-galactosidase assay.

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Rhizobium (Sinorhizobium)meliloti phn Genes: Characterization and Identification of Their Protein Products

Journal of Bacteriology ◽

10.1128/jb.181.2.389-395.1999 ◽

1999 ◽

Vol 181 (2) ◽

pp. 389-395 ◽

Cited By ~ 31

Author(s):

George F. Parker ◽

Timothy P. Higgins ◽

Timothy Hawkes ◽

Robert L. Robson

Keyword(s):

Escherichia Coli ◽

Sinorhizobium Meliloti ◽

Insertional Mutagenesis ◽

Biochemical Characterization ◽

Polyclonal Antibodies ◽

E Coli ◽

New Information ◽

Phosphorus Sources ◽

Carbon Phosphorus Lyase

ABSTRACT In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA fromRhizobium (Sinorhizobium) melilotithat contains homologues of the E. coli phnG, -H, -I, -J, and -Kgenes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phngenes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.

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