scholarly journals TRANSFORMASI PLASMID YANG MENGANDUNG GEN merB PADA Escherichia coli BL21(DE3)

PHARMACON ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 196
Author(s):  
Zefanya G Bernadus ◽  
Fatimawali Fatimawali ◽  
Beivy Kolondam
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Genetic Material ◽  
Luria Bertani ◽  
Agarose Gel ◽  
Bacterial Cells ◽  
E Coli ◽  
Escherichia Coli Bl21 ◽  
Plasmid Transformation ◽  
Merb Gene

ABSTRACTDNA transformation is one of the methods for inserting DNA into bacterial cells. The current transformation method is widely used to transfer plasmids containing genetic material. This study aims to evaluate the results of plasmid transformation containing merB gene in Escherichia coli BL21(DE3) bacteria. The stages of the research carried out were preceded by the microbiological identification of the E. coli BL21(DE3) bacteria used as hosts. Then the plasmid transformation containing merB gene into the E. coli BL21(DE3) host cell using the heat shock method was carried out. The transformation results were evaluated by observing at the presence of E. coli BL21(DE3) colonies on agar Luria Bertani (LB) media containing ampicillin antibiotics. Plasmids in E. coli BL21(DE3) were isolated and analyzed by 1% agarose gel electrophoresis. The results showed the success of the transformation indicated by the growth of E. coli BL21(DE3) bacteria in agar LB media containing ampicillin and the visualization on agarose gel resulted that the plasmid which carried the merB gene could be transformed in to the E. coli BL21(DE3) bacteria.Keywords : Plasmids, merB genes, heat shock, Escherichia coli BL21(DE3)ABSTRAKTransformasi DNA merupakan salah satu metode untuk memasukkan DNA ke dalam sel bakteri. Metode transformasi saat ini dipakai secara luas untuk mentransfer plasmid yang mengandung bahan genetika. Penelitian ini bertujuan untuk mengevaluasi hasil transformasi plasmid yang mengandung gen merB pada bakteri Escherichia coli BL21(DE3). Tahapan penelitian didahului dengan identifikasi secara mikrobiologi bakteri E. coli BL21(DE3) yang digunakan sebagai inang. Selanjutnya dilakukan transformasi plasmid yang mengandung gen merB kedalam sel inang E. coli BL21(DE3) menggunakan metode heat shock. Hasil transformasi dievaluasi dengan melihat adanya koloni E. coli BL21(DE3) pada media agar Luria Bertani (LB) yang mengandung antibiotik ampisilin. Plasmid pada E. coli BL21(DE3) diisolasi dan dianalisis dengan elektroforesis gel agarose 1%. Hasil penelitian menunjukkan keberhasilan transformasi dengan adanya pertumbuhan bakteri E. coli BL21(DE3) pada media LB yang mengandung ampisillin dan hasil visualisasi pada agarose gel terlihat bahwa plasmid yang membawa gen merB dapat ditransformasikan ke dalam bakteri E. coli BL21(DE3).Kata Kunci : Plasmid, gen merB, heat shock, Escherichia coli BL21(DE3)

scholarly journals TRANSFORMASI PLASMID YANG MENGANDUNG GEN merB PADA BAKTERI Escherichia coli TOP-10

PHARMACON ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 290
Author(s):  
Sarah Rotinsulu ◽  
Fatimawali Fatimawali ◽  
Trina E. Tallei
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Genetic Material ◽  
Transformation Method ◽  
Host Cells ◽  
Agarose Gel ◽  
Vector Plasmid ◽  
E Coli ◽  
Heat Shocks ◽  
Merb Gene

ABSTRACT DNA transformation is the process of inserting recombinant DNA into host cells via vector plasmid. The host cell that is often used is TOP-10 Escherichia coli. The transformation method is widely used to transfer plasmids containing genetic material. This study aimed to evaluate the results of plasmid transformation containing the merB gene in the Escherichia coli TOP-10 bacteria. This study initiated with identification of the microbiology of host cells to be used, namely Escherichia coli TOP-10. Escherichia coli TOP-10 host cells were made into competent cells by transforming plasmids containing merB gene into Escherichia coli TOP-10 host cells using the heat shock method. The transformation results were evaluated by observing at the growth of Escherichia coli TOP-10 colonies on agar LB media containing ampicillin antibiotics. Plasmids on Escherichia coli TOP-10 were isolated and analyzed by 1% agarose gel electrophoresis. The results showed that the transformation of plasmids containing merB in Escherichia coli TOP-10 bacteria was successfully carried out as indicated by the growth of Escherichia coli TOP-10 bacteria on LB media agar containing ampicillin and the visualization on agarose gel resulted that the plasmid which carried the merB gene could be transformed in to the E. coli TOP-10 bacteria cell. Keywords: Transformation, Plasmids, merB genes, heat shocks, E. coli TOP-10ABSTRAK Transformasi DNA merupakan proses memasukkan DNA kedalam sel bakteri. Metode transformasi dipakai secara luas untuk mantransfer plasmid yang mengandung bahan genetika. Penelitian ini bertujuan untuk mengevaluasi hasil transformasi plasmid yang mengandung gen merB pada bakteri Escherichia coli TOP-10. Penelitian ini didahului dengan identifikasi secara mikrobiologi bakteri Escherichia coli TOP-10. Bakteri Escherichia coli TOP-10 dibuat menjadi sel kompeten yang digunakan sebagai inang. Selanjutnya dilakukan transformasi plasmid yang mengandung gen merB kedalam sel inang E. coli TOP-10 menggunakan metode heatshock. Hasil transformasi dievaluasi dengan melihat adanya koloni E. coli TOP-10 pada media LB agar yang mengandung antibiotik ampisilin. Plasmid pada E. coli TOP-10 diisolasi dan dianalisis dengan elektroforesis gel  agarose 1%. Hasil penelitian menunjukkan bahwa transformasi plasmid yang mengandung gen merB pada bakteri Escherichia coli TOP-10 berhasil dilakukan, ditunjukkan dengan adanya pertumbuhan bakteri E. coli TOP-10 pada media LB agar yang mengangandung ampisilin dan hasil visualisasi pada agarose gel terlihat bahwa plasmid yang membawa gen merB dapat ditransformasikan ke dalam sel bakteri E. coli TOP-10. Kata kunci : Transformasi, Plasmid, gen merB, heatshock, E. coli TOP-10 

scholarly journals Heat shock transformation and expression of the plasmid containing cytolethal distending toxin of Campylobacter fetus subsp venerealis in Escherichia coli

2021 ◽  
Vol 888 (1) ◽  
pp. 012021
Author(s):  
N Herlina ◽  
N D Yanthi ◽  
R D Pratiwi ◽  
K S Dewi ◽  
F Setiyoningrum ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Luria Bertani ◽  
Protein Characterization ◽  
Bacterial Cells ◽  
Iptg Induction ◽  
E Coli ◽  
Sds Page ◽  
Fetus Subsp ◽  
Campylobacter Infection

Abstract The cytolethal distending toxins (cdt) is a multi-subunit toxin consisted of three subunit encoded cdtA, cdtB and cdtC. The cdt played an important role as a virulence factor of Campylobacter infection, including C. fetus subsp venerealis. The cdtA which responsible for binding the cdt to cell membrane, was cloned in plasmid expression and inserted into bacterial cells of Escherichia coli BL21(DE3). The research was conducted to evaluate the transformation using the heat shock method of a plasmid containing cdtA3 gene and the protein expression induced by various concentration of IPTG. Transformation was done using the heat shock method at 42oC for 90 second. Evaluation of the transformation was observed on the presence of E. coli BL21(DE3) colonies on Luria Bertani agar containing Ampicillin antibiotic with 100 µg/mL dosage. The recombinant protein was expressed using IPTG-induction with various concentration (0.1mM, 0.25mM, 0.5mM, 0.75mM and 1 mM). The result showed that the transformation and IPTG-induction 0.1 mM produced higher concentration of protein than other concentration applied. The protein characterization was observed with SDS PAGE and cdtA3 protein was detected on 23,4 kDa.

Increased Transcription of the Phosphate-Specific Transport System of Escherichia coli O157:H7 after Exposure to Sodium Benzoate

2010 ◽  
Vol 73 (5) ◽  
pp. 819-824 ◽  
Author(s):  
FAITH J. CRITZER ◽  
DORIS H. D'SOUZA ◽  
ARNOLD M. SAXTON ◽  
DAVID A. GOLDEN
Keyword(s):  
Escherichia Coli ◽  
Sodium Benzoate ◽  
Efflux Pump ◽  
Transcriptional Response ◽  
Luria Bertani ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
E Coli ◽  
Specific Transport ◽  
Pst System

Sodium benzoate is a widely used food antimicrobial in drinks and fruit juices. A microarray study was conducted to determine the transcriptional response of Escherichia coli O157:H7 to 0.5% (wt/vol) sodium benzoate. E. coli O157:H7 grown in 150 ml of Luria-Bertani broth was exposed to 0% (control) and 0.5% sodium benzoate. Each treatment was duplicated and sampled at 0 (immediately after exposure), 5, 15, 30, and 60 min. Total RNA was extracted and analyzed with E. coli 2.0 Gene Chips. Significant ontology categories affected by sodium benzoate exposure were determined with JProGO software. The phosphate-specific transport (Pst) system transports inorganic phosphate into bacterial cells, under phosphate-limited conditions. The Pst system was found to be highly upregulated. Increased expression of the Pst system was observed after the short 5 min of exposure to sodium benzoate; pstS, pstA, pstB, and pstC genes were upregulated more than twofold (linear scale) at 5, 15, 30, and 60 min. Increased expression of several other efflux systems, such as AcrAB-TolC, was also observed. The Pst system may act as an efflux pump under these stress-adapted conditions, as well as increase transport of phosphorus to aid in DNA, RNA, ATP, and phospholipid production. Understanding adaptations of Escherichia coli O157:H7 under antimicrobial exposure is essential to better understand and implement methods to inhibit or control its survival in foods.

scholarly journals Stability of plasmid pBR322 within Escherichia coli cells

2021 ◽  
Vol 6 (1) ◽  
pp. 17-19
Author(s):  
Tahsin Tabassum ◽  
Tasmin Tabassum ◽  
Nafisa Tabassum ◽  
Syeda Muntaka Maniha ◽  
Rashed Noor
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Heat Shock ◽  
Plasmid Stability ◽  
Bacterial Cells ◽  
Plasmid Pbr322 ◽  
E Coli ◽  
Replica Plating ◽  
Escherichia Coli Cells ◽  
Pbr322 Plasmid

nsertion of plasmids into the bacterial cells is of great significance especially in course of the transfer of drug resistance, virulence and other traits. Retention of plasmids within the host bacteria is therefore an important factor for bacterial homeostasis. Current study inferred the pBR322 plasmid stability within the Escherichia coli competent cells. The calcium chloride heat shock method was used for the transformation purpose. The plasmid retention phenomenon was assessed through the replica plating. The results positively showed the plasmid retention within E. coli.

scholarly journals Antimicrobial Resistance in Escherichia coli

2021 ◽  
Author(s):  
Waheed Ullah ◽  
Shandana Ali
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Genetic Material ◽  
Resistance Pattern ◽  
Bacterial Cells ◽  
Sources Of Resistance ◽  
E Coli ◽  
Sub Saharan ◽  
Different Strains

The ability of microbes to resist or neutralize the action of drugs that have been used against microbes is considered as antimicrobial resistance (AMR). AMR among different strains of Escherichia coli is considered as a major threat to public health. Drug-resistant in E. coli is found predominantly in the hospital sittings, in the community, and surrounding environment. It has adopted different defensive strategies to minimize the effects of drugs. Extended-spectrum β-lactamase (ESBL), fluoroquinolones, and carbapenemases have been considered as strong resistance strategies being present in most of resistant bacterial strains. Mobile genetic elements (MGEs) have the major contribution in the transfer of resistance genes in between or among bacterial cells. Plasmids are normally present in most of resistant strains, helping in the transfer of genetic material between bacterial cells. Transposons another MGEs, are being considered as one of the major sources of resistance transmission. Collectively, MGEs play an important role in facilitating in exchange, acquisition, and dissemination of resistance genes. Resistance in E. coli has been reported worldwide and there is variation in its resistance pattern. CTX-M ESBLs, carbapenems, colistin-resistant, and ST-131 E. coli resistant clones are considered the most dominant phenotypes. The aforesaid resistant variants are predominantly found in densely populated regions, Sub-Saharan Africa, China, and South Asian countries.

scholarly journals Antimicrobial activity of Syzygium aromaticum L. essential oil on extended-spectrum beta-lactamases-producing Escherichia coli

2020 ◽  
Vol 44 (1) ◽  
Author(s):  
E. L. Mejía-Argueta ◽  
J. G. Santillán-Benítez ◽  
M. M. Canales-Martinez ◽  
A. Mendoza-Medellín
Keyword(s):  
Escherichia Coli ◽  
Essential Oil ◽  
Genetic Material ◽  
Gastrointestinal Diseases ◽  
Syzygium Aromaticum ◽  
Antibiotic Resistant ◽  
Plant Essential Oils ◽  
E Coli ◽  
Extended Spectrum ◽  
Tract Infections

Abstract Background To test the antimicrobial potential of clove essential oil that has been less investigated on antimicrobial-resistant organisms (extended-spectrum β-lactamase-ESBL-producing Escherichia coli), we collected 135 ESBL-producing Escherichia coli strains given that E. coli is the major organism increasingly isolated as a cause of complicated urinary and gastrointestinal tract infections, which remains an important cause of therapy failure with antibiotics for the medical sector. Then, in this study, we evaluated the relationship between the antibacterial potential activity of Syzygium aromaticum essential oil (EOSA) and the expression of antibiotic-resistant genes (SHV-2, TEM-20) in plasmidic DNA on ESBL-producing E. coli using RT-PCR technique. Results EOSA was obtained by hydrodistillation. Using Kirby-Baüer method, we found that EOSA presented a smaller media (mean = 15.59 mm) in comparison with chloramphenicol (mean = 17.73 mm). Thus, there were significant differences (p < 0.0001). Furthermore, EOSA had an antibacterial activity, particularly on ECB132 (MIC: 10.0 mg/mL and MBC: 80.0 mg/mL), and a bacteriostatic effect by bactericidal kinetic. We found that the expression of antibiotic-resistant gene blaTEM-20 was 23.52% (4/17 strains) and no expression of blaSHV-2. EOSA presented such as majority compounds (eugenol, caryophyllene) using the GC–MS technique. Conclusions Plant essential oils and their active ingredients have potentially high bioactivity against a different target (membranes, cytoplasm, genetic material). In this research, EOSA might become an important adjuvant against urinary and gastrointestinal diseases caused by ESBL-producing E. coli.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals A Pathoadaptive Deletion in an Enteroaggregative Escherichia coli Outbreak Strain Enhances Virulence in a Caenorhabditis elegans Model

2010 ◽  
Vol 78 (9) ◽  
pp. 4068-4076 ◽  
Author(s):  
Jennifer Hwang ◽  
Lisa M. Mattei ◽  
Laura G. VanArendonk ◽  
Philip M. Meneely ◽  
Iruka N. Okeke
Keyword(s):  
Escherichia Coli ◽  
Caenorhabditis Elegans ◽  
Epithelial Cells ◽  
Genetic Material ◽  
Decarboxylase Activity ◽  
Multicopy Plasmid ◽  
Lysine Decarboxylase ◽  
Outbreak Strain ◽  
E Coli ◽  
C Elegans

ABSTRACT Enteroaggregative Escherichia coli (EAEC) strains are important diarrheal pathogens. EAEC strains are defined by their characteristic stacked-brick pattern of adherence to epithelial cells but show heterogeneous virulence and have different combinations of adhesin and toxin genes. Pathoadaptive deletions in the lysine decarboxylase (cad) genes have been noted among hypervirulent E. coli subtypes of Shigella and enterohemorrhagic E. coli. To test the hypothesis that cad deletions might account for heterogeneity in EAEC virulence, we developed a Caenorhabditis elegans pathogenesis model. Well-characterized EAEC strains were shown to colonize and kill C. elegans, and differences in virulence could be measured quantitatively. Of 49 EAEC strains screened for lysine decarboxylase activity, 3 tested negative. Most notable is isolate 101-1, which was recovered in Japan, from the largest documented EAEC outbreak. EAEC strain 101-1 was unable to decarboxylate lysine in vitro due to deletions in cadA and cadC, which, respectively, encode lysine decarboxylase and a transcriptional activator of the cadAB genes. Strain 101-1 was significantly more lethal to C. elegans than control strain OP50. Lethality was attenuated when the lysine decarboxylase defect was complemented from a multicopy plasmid and in single copy. In addition, restoring lysine decarboxylase function produced derivatives of 101-1 deficient in aggregative adherence to cultured human epithelial cells. Lysine decarboxylase inactivation is pathoadapative in an important EAEC outbreak strain, and deletion of cad genes could produce hypervirulent EAEC lineages in the future. These results suggest that loss, as well as gain, of genetic material can account for heterogeneous virulence among EAEC strains.

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

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
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.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

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