plasmid isolation
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Author(s):  
Daniel Lantang ◽  
Arsyam Mawardi

This research aims to analyze the level of similarity and diversity among local isolates of B. thuringiensis Auky Island Padaido District in Biak Numfor Regency with NCBI gene bank base, the basis of which is to obtain B. thuringiensis isolates from jayapura local isolates that can act as controllers of Anopheles mosquito larvae. Several steps in the research are 16s gene amplification, PCR product purification, cloning using pTA2 vectors and transformation into competent E. coli Zymo 5α cells, confirmation with PCR colonies, recombinant plasmid isolation, sequencing analysis and phylogenetic tree construction. The isolates of ABNP8, ABNP9, ABNP11, ABNP12 and ABNP18 have been detected as local isolates from in Auky Island Padaido District in Biak Numfor Papua Regency that have great potential as bioinsecticides, and capable of controlling and killing Anopheles mosquito larvae. Of the five isolates, ABNP8 isolates had unique diversity and characteristics and were different from the four other isolates. Based on the similarity analysis in the MEGA7 program, the similarity rate reached 84%. Its diversity can be seen from the uniqueness of the sequence and its position in different branching dendrograms.


2018 ◽  
Vol 10 (1) ◽  
Author(s):  
Arsyam Mawardi ◽  
Leonardo E. Aisoi ◽  
Paula N. Lefaan

Cloning gene involves the construction of a recombinant plasmid that inserted in a competent cell. On the other hand, genetic engineering requires bioinformatic analysis to be converted into tabulation and data interpretation. The study, titled "cloning block 2 MSP1 gene of Plasmodium falciparum isolate Jayapura city and bioinformatics analysis" is aimed to improve the technique of cloning the MSP1 gene of P. falciparum, initiated the creation of DH5α competent cells, ligations and transformations, plasmid isolation, confirmation the recombinant plasmid and able to perform bioinformatics analysis and construct phylogenetic tree. This study began with the manufacture of E. coli DH5α competent cells, MSP1 gene ligation in pJET1.2/blunt vector and transformation by using the heat shock transformation method, plasmid isolation of alkali lysis method, then plasmid confirmed by PCR and sequencing method, further sequence analysis and phylogenetic tree construction. The results showed that confirmation of MSP1 gene presence in pJET1.2/blunt with PCR was successful. From a total of 4 positive colonies grown in liquid culture, then isolated plasmid and confirmed with PCR obtained electroferogram bands with a size about 1049 bp indicates the presence of MSP1 gene in plasmid. Based on the results, cloning of MSP1 gene using pJET1.2/blunt cloning vector and competent cell E. coli DH5α has been successfully performed. Bioinformatics analysis of sequencing result and phylogenetic tree were constructed successfully with 2 clusters isolate of malaria patients from Jayapura city. Key words: Bioinformatics, cloning gene, heat shock transformation, MSP1, P. falciparum.


2016 ◽  
Vol 4 (2) ◽  
pp. 321
Author(s):  
Ebakota Daniel ◽  
Osarueme Osazee ◽  
Frances Olisaka ◽  
Jocelyn Aibangbee ◽  
Panmwa GALAU ◽  
...  

The indiscriminate use of antibiotics by individuals as well as in food production has been tagged one of the major reasons for the spread of antibiotic resistance in pathogens. Thus, there is a concern that foodborne bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses with food. This study aimed at determining the antibiotic susceptibility, plasmid isolation and curing of foodborne bacteria isolated from ready to eat (RTE) foods and salads in eating centers at the Benson Idahosa University, Benin City. Isolates were Enterobacter aerogenes, Escherichia coli, Staphylococcus aureus, Bacillus spp., Micrococcus sp. and Salmonella sp with S. aureus occurring most frequently. Total resistance to cefuroxime and augmentin as well as considerable resistance to ceftazidime and cefixime were observed in all isolates in antimicrobial susceptibility tests were done on Mueller-Hinton agar. Relative sensitivity to gentamicin, ofloxacin, nitrofurantoin and ciprofloxacin were observed. Plasmid profiling indicated that all isolates possess plasmids ranging from 100 bp to 1 kbp. Plasmid curing using sodium dodecyl sulfate (SDS) improved the sensitivity of isolates to antibiotics they were previously sensitive to but most isolates remained resistance to ceftazidime, cefuroxime, cefixime, and augmentin. This study shows that foodborne bacteria can possess and possibly transfer persistent antibiotic resistance plasmids thus calling for more caution in the use of antibiotics in food production and reduced antibiotics abuse. Further research is currently ongoing to cure the isolates of all plasmids and to elucidate how these plasmids are being transferred.


2016 ◽  
Vol 18 (2) ◽  
pp. 61
Author(s):  
Sutanti Sutanti ◽  
Novi Megawati ◽  
Sugiyo H. Pranoto ◽  
Ratu Siti Aliah

DNA vaccine Streptococcus iniae is a third generation of vaccines based on the gene encoding a vaccine antigen. Pgm is DNA-binding protein that activates expression of several important virulence gene, including those encoding polysaccharide capsule. The pgm gene controlled by MBA promoter has constructed successfully as a candidate of DNA vaccine to against S. iniae infection in Nile Tilapia. The goals of this study were to production and application of DNA Vaccine S. iniae to increase the nile tilapia immunity.  Vaccine production was using plasmid isolation method from Escherichia coli dH5α containing pMBA-pgm. Vaccine verification was was accomplished by PCR using pgm gene S. iniae Fstrep and Rstrep specific primers, which revealed the 1,713 bp PCR product. Application of DNA vaccine was using 25, 50, 75, and 100ng/ml dosage with intraperitonial injection method .The challenge test was using 108 cfu/ml density of S. iniae. Observation test parameter were survival rate, relative percent survival, mean time to death, pgm gene, and organ histology such as liver, eyes, brain, spleen and kidney. Vaccine production was succeed using plasmid isolation method and containing pgm gene revealed 1713bp. the application of DNA vaccine was optimum at 50ng/ml dosage with SR value 96.667%, RPS value 88,461% and MTD value 4,6 days. The eyes histology shows opacity and exopthalmia, the other organ such as liver, spleen and kidney was necrosis. The DNA vaccine was optimum at 50ng/ml dosage.  


2016 ◽  
Vol 10 (02) ◽  
pp. 183-187 ◽  
Author(s):  
Camille-Ann Thoms-Rodriguez ◽  
Tony Mazzulli ◽  
Nicole Christian ◽  
Barbara M Willey ◽  
David A Boyd ◽  
...  

Introduction: The global dissemination of the New Delhi metallo-beta-lactamase (NDM) gene among certain strains of bacteria has serious implications since the infections caused by such organisms pose a therapeutic challenge. Although the NDM gene has been detected in various parts of the world, this is the first report of its detection in the English-speaking Caribbean. The NDM producing Klebsiella pneumoniae was isolated from an Indian patient who had recently relocated to Jamaica. Methodology: Identification and susceptibility testing of the K. pneumoniae isolate was performed using the Vitek 2 automated system) in keeping with Clinical and Laboratory Standards Institute (CLSI) standards. It was identified as a metallobetalactamase producer using the Rosco KPC+MBL kit. Genotypic screening for common betalactamase (including carbapenemase) genes, was carried out  using two multiplex PCRs: one for SHV-, TEM-, CTX-M-, OXA-1-, and CMY-2-types, and one for VIM-, KPC-, IMP-, OXA-48, GES-, and NDM-types. Strain typing was conducted by pulsed-field gel electrophoresis (PFGE) using XbaI and multi-locus sequencing (MLS). Plasmid isolation and analysis was also performed. Results: K. pneumoniae (N11-02395), not previously associated with the dissemination of the NDM in India, Sweden or the UK, was found to harbor the NDM-1 gene on plasmid pNDM112395. Conclusion: The identification of the NDM-1 gene underscores the need for effective surveillance and infection control measures to identify and prevent spread of multidrug resistant Gram negative bacilli. Strict infection control measures implemented for this patient helped to prevent the spread of this organism to other patients.


2015 ◽  
Vol 2 (2) ◽  
pp. 60
Author(s):  
Dudi Hardianto ◽  
Alfik Indarto ◽  
Nurtjahjo Dwi Sasongko

Plasmids are extra chromosomal molecules of DNA that replicate autonomously and found in prokaryote and eukaryote cells. There are a number of methods that are used to isolate plasmids, such as alkaline lysis, boiling lysis, using cesium chloride, and chromatography. Amongst the disadvantages in plasmid isolation methods are lengthy time especially when handling a large number of samples, high cost, and low purity. Alkaline lysis is the most popular for plasmid isolation because of its simplicity, relatively low cost, and reproducibility. This method can be accomplished in 50 minutes to one hour. In this research, the alkaline lysis method was developed to obtain suitable plasmid for applications in a molecular biology laboratory. The aim of this research was to reduce contaminants and improve yield of plasmid. The result of isolation of pICZA plasmid in Escherichia coli gave the concentration of 3.3 to 3.8 µg/µL with the purity of 1.99.Keywords: Plasmid isolation, pICZ A, Escherichia coli, rapid, alkaline lysis  ABSTRAKPlasmid merupakan molekul DNA ekstrakromosomal yang bereplikasi secara mandiri dan ditemukan dalam sel prokariot dan eukariot. Banyak metode yang digunakan untuk isolasi plasmid, seperti: lisis alkali, lisis dengan pemanasan, penggunaan sesium klorida, dan kromatografi. Kelemahan beberapa metode isolasi DNA adalah waktu isolasi yang lama terutama saat isolasi plasmid dalam jumlah banyak, mahal dan kemurniannya yang rendah. Metode lisis alkali merupakan metode yang sangat umum untuk isolasi plasmid karena mudah dilakukan, relatif murah, dan reprodusibilitas. Metode ini dapat dilakukan dalam 50 menit sampai 1 jam. Pada penelitian ini dikembangkan metode lisis alkali untuk memperoleh plasmid yang sesuai untuk penggunaan di laboratorium biologi molekuler. Tujuan dari penelitian ini adalah untuk mengurangi jumlah kontaminan dan meningkatkan konsentrasi plasmid. Hasil isolasi plasmid pICZA dalam Escherichia coli mempunyai konsentrasi antara 3,3 sampai 3,8 µg/µL dan kemurniannya 1,99.Kata Kunci: Isolasi plasmid, pICZ A, Escherichia coli, cepat, lisis alkali


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