Molecular cloning of polyoma virus DNA in Escherichia coli: lambda phage vector system

Science ◽  
1979 ◽  
Vol 203 (4383) ◽  
pp. 887-892 ◽  
Author(s):  
H. Chan ◽  
M. Israel ◽  
C. Garon ◽  
W. Rowe ◽  
M. Martin
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Polyoma Virus ◽  
Vector System ◽  
Lambda Phage

Molecular cloning of polyoma virus DNA in Escherichia coli: plasmid vector system

Science ◽  
1979 ◽  
Vol 203 (4383) ◽  
pp. 883-887 ◽  
Author(s):  
M. Israel ◽  
H. Chan ◽  
W. Rowe ◽  
M. Martin
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Plasmid Vector ◽  
Polyoma Virus ◽  
Vector System ◽  
Coli Plasmid

Construction of a vector system for molecular cloning in Bacillus subtilis and Escherichia coli

2005 ◽  
Vol 39 (2) ◽  
pp. 306-309 ◽  
Author(s):  
A. V. Lagodich ◽  
E. A. Cherva ◽  
Ya. V. Shtaniuk ◽  
V. A. Prokulevich ◽  
Yu. K. Fomichev ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Molecular Cloning ◽  
Vector System

scholarly journals Molecular Cloning of Selected Regions of Catalytic Domain of CtsK Gene in Escherichia coli – A Feasibility Study

2021 ◽  
pp. 11-35
Author(s):  
Hasanka Madubashetha ◽  
Ruwini Cooray ◽  
P. D. S. U. Wickramasinghe ◽  
Lakshan Warnakula ◽  
Nimali De Silva
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Catalytic Domain ◽  
Expression System ◽  
Cathepsin K ◽  
Restriction Enzymes ◽  
Vector System ◽  
Restriction Enzyme Digestion ◽  
Healthy Human ◽  
Band Size

Cathepsin K (CatK), encoded by CtsK gene in human, is involved in bone remodeling through ossification. The objective of the work conducted here was to express catalytic domains of CtsK gene in bacterial expression system as an initial step, facilitating recombinant production of human CatK for downstream applications in pharmacology. Four healthy human blood samples were collected. Genomic DNA was extracted using FlexiGene® whole blood DNA extraction kit. Upon quantification of DNA through NanodropTM spectrophotometer, sufficient quantity and quality was observed. CtsK gene was amplified by Polymerase Chain Reaction (PCR) using two pairs of primers tagged with restriction endonuclease sites of Sal1 and HindIII facilitating molecular cloning and visualized by Agarose Gel Electrophoresis (AGE). Two different bands of size 545bp and 265bp were observed. The bands were dissected and gel purified using GenaxxonTM gel purification kit and sequentially double digested by restriction enzymes; SalI and HindIII.  Vector PBS was also subjected to sequential double digestion using same enzymes and visualized via AGE. Double digested insert of size 265bp and vector were ligated using T4 DNA Ligase (all enzymes from PromegaTM). On another trail, ligation of the PCR product with band size 265bp to pGEM-TTM easy vector system (from PromegaTM) was also done and transformed to Top10 Escherichia coli competent cells for expression separately. Cells were grown in LB media in presence of XGAL, IPTG and Ampicillin and transformed cells were screened. In the restriction enzyme digestion and ligation setup, since the insert and vector were both double digested, it is confirmed that white colonies obtained were Escherichia coli cells were transformed with the desired recombinant vector and is therefore confirmatory. In the case of pGEM-TTM ligation, a colony PCR was done using the white colonies obtained and product size was confirmed via AGE. In conclusion, the objective of study was successfully achieved, by expressing a catalytic domain of CtsK. Developments and improvements could be made for expression of entire CatK gene and downstream production of the Cathepsin K protein for effective therapeutic purpose.

Molecular cloning of polyoma virus DNA in Escherichia coli: oncogenicity testing in hamsters

Science ◽  
1979 ◽  
Vol 205 (4411) ◽  
pp. 1140-1142 ◽  
Author(s):  
M. Israel ◽  
H. Chan ◽  
M. Martin ◽  
W. Rowe
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Polyoma Virus

scholarly journals Further tests of a recombination model in which chi removes the RecD subunit from the RecBCD enzyme of Escherichia coli.

Genetics ◽  
1990 ◽  
Vol 126 (3) ◽  
pp. 519-533 ◽  
Author(s):  
F W Stahl ◽  
L C Thomason ◽  
I Siddiqi ◽  
M M Stahl
Keyword(s):  
Escherichia Coli ◽  
Dna Sequence ◽  
Dna Packaging ◽  
Lambda Phage ◽  
Recombination Model ◽  
Phage Types ◽  
Point Of Entry ◽  
Reciprocal Recombination ◽  
Recbcd Enzyme ◽  
Lambda Dna

Abstract When one of two infecting lambda phage types in a replication-blocked cross is chi + and DNA packaging is divorced from the RecBCD-chi interaction, complementary chi-stimulated recombinants are recovered equally in mass lysates only if the chi + parent is in excess in the infecting parental mixture. Otherwise, the chi 0 recombinant is recovered in excess. This observation implies that, along with the chi 0 chromosome, two chi + parent chromosomes are involved in the formation of each chi + recombinant. The trimolecular nature of chi +-stimulated recombination is manifest in recombination between lambda and a plasmid. When lambda recombines with a plasmid via the RecBCD pathway, the resulting chromosome has an enhanced probability of undergoing lambda x lambda recombination in the interval into which the plasmid was incorporated. These two observations support a model in which DNA is degraded by Exo V from cos, the sequence that determines the end of packaged lambda DNA and acts as point of entry for RecBCD enzyme, to chi, the DNA sequence that stimulates the RecBCD enzyme to effect recombination. The model supposes that chi acts by ejecting the RecD subunit from the RecBCD enzyme with two consequences. (1) ExoV activity is blocked leaving a highly recombinagenic, frayed duplex end near chi, and (2) as the enzyme stripped of the RecD subunit travels beyond chi it is competent to catalyze reciprocal recombination.

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