Displaying publications 61 - 80 of 224 in total

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  1. Auxotypes and serogroups of tetracycline-resistant Neisseria gonorrhoeae isolated in Malaysia
    Koay AS, My R, Cheong YM
    J Clin Microbiol, 1996 Jul;34(7):1863-5.
    PMID: 8784614
    Between 1992 and 1994, 253 tetracycline-resistant Neisseria gonorrhoeae (TRNG) strains were isolated and characterized by auxotype and serogroup (A/S) classes to study TRNG prevalence in different years. TRNG accounted for 28.1, 42.5, and 51.9% of the strains isolated in 1992, 1993, and 1994, respectively, showing a significant increase in each successive year (chi square = 26.7, P < 0.001). There was no significant increase in penicillinase-producing TRNG, which accounted for 53.1, 53.8, and 63.2% of the TRNG isolates. The 253 TRNG isolates belonged to 53 A/S classes. Eighteen A/S classes not observed in 1992 were detected in 1993, and 11 A/S classes not observed in 1992 and 1993 were isolated in 1994, indicating dissemination of the tetracycline resistance gene among the N. gonorrhoeae strains in Malaysia. Its emergence and subsequent rapid spread are alarming. The plasmid is capable of self-transfer (S.A. Morse, S.R. Johnson, J.W. Biddle, and M.C. Roberts, J. Infect. Dis. 155:819-822, 1987), allowing further dissemination of tetracycline resistance.
    Matched MeSH terms: Plasmids/genetics
  2. Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan
    Kawalek MD, Benjamin S, Lee HL, Gill SS
    Appl Environ Microbiol, 1995 Aug;61(8):2965-9.
    PMID: 7487029
    A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.
    Matched MeSH terms: Plasmids/genetics
  3. Recombineering linear BACs
    Chen Q, Narayanan K
    Methods Mol Biol, 2015;1227:27-54.
    PMID: 25239740 DOI: 10.1007/978-1-4939-1652-8_2
    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
    Matched MeSH terms: Plasmids/metabolism; Plasmids/chemistry
  4. Dynamics of PEGylated-dextran-spermine nanoparticles for gene delivery to leukemic cells
    Amini R, Jalilian FA, Abdullah S, Veerakumarasivam A, Hosseinkhani H, Abdulamir AS, et al.
    Appl Biochem Biotechnol, 2013 Jun;170(4):841-53.
    PMID: 23615733 DOI: 10.1007/s12010-013-0224-0
    Leukemic cells are hard-to-transfect cell lines. Many transfection reagents which can provide high gene transfer efficiency in common adherent cell lines are not effective to transfect established blood cell lines or primary leukemic cells. This study aims to examine a new class of cationic polymer non-viral vector, PEGylated-dextran-spermine (PEG-D-SPM), to determine its ability to transfect the leukemic cells. Here, the optimal conditions of the complex preparation (PEG-D-SPM/plasmid DNA (pDNA)) were examined. Different weight-mixing (w/w) ratios of PEG-D-SPM/pDNA complex were prepared to obtain an ideal mixing ratio to protect encapsulated pDNA from DNase degradation and to determine the optimal transfection efficiency of the complex. Strong complexation between polymer and pDNA in agarose gel electrophoresis and protection of pDNA from DNase were detected at ratios from 25 to 15. Highest gene expression was detected at w/w ratio of 18 in HL60 and K562 cells. However, gene expression from both leukemic cell lines was lower than the control MCF-7 cells. The cytotoxicity of PEG-D-SPM/pDNA complex at the most optimal mixing ratios was tested in HL60 and K562 cells using MTS assay and the results showed that the PEG-D-SPM/pDNA complex had no cytotoxic effect on these cell lines. Spherical shape and nano-nature of PEG-D-SPM/pDNA complex at ratio 18 was observed using transmission electron microscopy. As PEG-D-SPM showed modest transfection efficiency in the leukemic cell lines, we conclude that further work is needed to improve the delivery efficiency of the PEG-D-SPM.
    Matched MeSH terms: Plasmids/genetics; Plasmids/chemistry
  5. Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmid-Mg/Al-LDH nanobiocomposite material as a vector for gene expression
    Masarudin MJ, Yusoff K, Rahim RA, Hussein MZ
    Nanotechnology, 2009 Jan 28;20(4):045602.
    PMID: 19417322 DOI: 10.1088/0957-4484/20/4/045602
    The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO(3)(-) layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg(2+) to Al(3+) molar ratio R(i) = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 A in LDH to 42 A was observed in the resulting nanobiocomposite, indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD) results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO(3) after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself.
    Matched MeSH terms: Plasmids/genetics; Plasmids/chemistry*
  6. Fulltext Engineering an L-cell line that expresses insulin under the control of the glucagon-like peptide-1 promoter for diabetes treatment
    Rasouli M, Ahmad Z, Omar AR, Allaudin ZN
    BMC Biotechnol, 2011 Nov 03;11:99.
    PMID: 22047106 DOI: 10.1186/1472-6750-11-99
    BACKGROUND: Diabetes mellitus is a complicated disease with a pathophysiology that includes hyperinsulinemia, hyperglycemia and other metabolic impairments leading to many clinical complications. It is necessary to develop appropriate treatments to manage the disease and reduce possible acute and chronic side effects. The advent of gene therapy has generated excitement in the medical world for the possible application of gene therapy in the treatment of diabetes. The glucagon-like peptide-1 (GLP-1) promoter, which is recognised by gut L-cells, is an appealing candidate for gene therapy purposes. The specific properties of L-cells suggest that L-cells and the GLP-1 promoter would be useful for diabetes therapy approaches.

    RESULTS: In this study, L-cells were isolated from a primary intestinal cell line to create suitable target cells for insulin expression studies. The isolated cells displayed L-cell properties and were therefore used as an L-cell surrogate. Next, the isolated L-cells were transfected with the recombinant plasmid consisting of an insulin gene located downstream of the GLP-1 promoter. The secretion tests revealed that an increase in glucose concentration from 5 mM to 25 mM induced insulin gene expression in the L-cells by 2.7-fold. Furthermore, L-cells quickly responded to the glucose stimulation; the amount of insulin protein increased 2-fold in the first 30 minutes and then reached a plateau after 90 minutes.

    CONCLUSION: Our data showed that L-cells efficiently produced the mature insulin protein. In addition, the insulin protein secretion was positively regulated with glucose induction. In conclusion, GLP-1 promoter and L-cell could be potential candidates for diabetes gene therapy agents.

    Matched MeSH terms: Plasmids/genetics*; Plasmids/chemistry
  7. Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative γ-butyrolactone receptor protein from Streptomyces fradiae
    Mohd-Sharif N, Shaibullah S, Givajothi V, Tan CS, Ho KL, Teh AH, et al.
    Acta Crystallogr F Struct Biol Commun, 2017 02 01;73(Pt 2):109-115.
    PMID: 28177322 DOI: 10.1107/S2053230X17001212
    TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
    Matched MeSH terms: Plasmids/metabolism; Plasmids/chemistry
  8. Fulltext Evolutionary study of Yersinia genomes deciphers emergence of human pathogenic species
    Tan SY, Tan IK, Tan MF, Dutta A, Choo SW
    Sci Rep, 2016 10 31;6:36116.
    PMID: 27796355 DOI: 10.1038/srep36116
    On record, there are 17 species in the Yersinia genus, of which three are known to be pathogenic to human. While the chromosomal and pYV (or pCD1) plasmid-borne virulence genes as well as pathogenesis of these three species are well studied, their genomic evolution is poorly understood. Our study aims to predict the key evolutionary events that led to the emergence of pathogenic Yersinia species by analyzing gene gain-and-loss, virulence genes, and "Clustered regularly-interspaced short palindromic repeats". Our results suggest that the most recent ancestor shared by the human pathogenic Yersinia was most probably an environmental species that had adapted to the human body. This might have led to ecological specialization that diverged Yersinia into ecotypes and distinct lineages based on differential gene gain-and-loss in different niches. Our data also suggest that Y. pseudotuberculosis group might be the donor of the ail virulence gene to Y. enterocolitica. Hence, we postulate that evolution of human pathogenic Yersinia might not be totally in parallel, but instead, there were lateral gene transfer events. Furthermore, the presence of virulence genes seems to be important for the positive selection of virulence plasmid. Our studies provide better insights into the evolutionary biology of these bacteria.
    Matched MeSH terms: Plasmids/genetics; Plasmids/metabolism
  9. Fulltext Occurrence and Antibiotic Resistance of Vibrio parahaemolyticus from Shellfish in Selangor, Malaysia
    Letchumanan V, Pusparajah P, Tan LT, Yin WF, Lee LH, Chan KG
    Front Microbiol, 2015;6:1417.
    PMID: 26697003 DOI: 10.3389/fmicb.2015.01417
    High consumer demand for shellfish has led to the need for large-scale, reliable shellfish supply through aquaculture or shellfish farming. However, bacterial infections which can spread rapidly among shellfish poses a major threat to this industry. Shellfish farmers therefore often resort to extensive use of antibiotics, both prophylactically and therapeutically, in order to protect their stocks. The extensive use of antibiotics in aquaculture has been postulated to represent a major contributing factor in the rising incidence of antimicrobial resistant pathogenic bacteria in shellfish. This study aimed to investigate the incidence of pathogenic Vibrio parahaemolyticus and determine the antibiotic resistance profile as well as to perform plasmid curing in order to determine the antibiotic resistance mediation. Based on colony morphology, all 450 samples tested were positive for Vibrio sp; however, tox-R assay showed that only 44.4% (200/450) of these were V. parahaemolyticus. Out of these 200 samples, 6.5% (13/200) were trh-positive while none were tdh-positive. Antibiotic resistance was determined for all V. parahaemolyticus identified against 14 commonly used antibiotics and the multiple antibiotic resistance index (MAR) was calculated. The isolates demonstrated high resistance to several antibiotics tested- including second and third-line antibiotics- with 88% resistant to ampicillin, 81% to amikacin,70.5% to kanamycin, 73% to cefotaxime, and 51.5% to ceftazidime. The MAR index ranged from 0.00 to 0.79 with the majority of samples having an index of 0.36 (resistant to five antibiotics). Among the 13 trh-positive strains, almost 70% (9/13) demonstrated resistance to 4 or more antibiotics. Plasmid profiling for all V. parahaemolyticus isolates revealed that 86.5% (173/200) contained plasmids - ranging from 1 to 7 plasmids with DNA band sizes ranging from 1.2 kb to greater than 10 kb. 6/13 of the pathogenic V. pathogenic strains contained plasmid. After plasmid curing, the plasmid containing pathogenic strains isolated in our study have chromosomally mediated ampicillin resistance while the remaining resistance phenotypes are plasmid mediated. Overall, our results indicate that while the incidence of pathogenic V. parahaemolyticus in shellfish in Selangor still appears to be at relatively reassuring levels, antibiotic resistance is a real concern and warrants ongoing surveillance.
    Matched MeSH terms: Plasmids
  10. Fulltext Development of Transgenic Papaya through Agrobacterium-Mediated Transformation
    Azad MA, Rabbani MG, Amin L, Sidik NM
    Int J Genomics, 2013;2013:235487.
    PMID: 24066284 DOI: 10.1155/2013/235487
    Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi.
    Matched MeSH terms: Plasmids
  11. Molecular delivery of plasmids for genetic vaccination
    Mazid R, Tan MX, Danquah MK
    Curr Pharm Biotechnol, 2013;14(6):615-22.
    PMID: 24016267
    Plasmid vaccination is a smart gene delivery application mostly achieved through the utilisation of viral or copolymeric systems as surrogated carriers in micro or nano formulations. A common polymeric protocol for plasmid vaccine formulation, which as somewhat been successful, is via the complexation of the DNA molecules with a cationic polymer, and encapsulating in a vehicular carrier polymer. Even though plasmid vaccination research has not witnessed the much anticipated success, due a number of cellular and physicochemical reasons, application of copolymeric carriers with tight functionalities is a promising strategy to optimally deliver the DNA molecules; in view of the available chemistries and physical properties that could be tuned to enable enhanced targeted delivery, uptake and specific transfection. This also enables the targeting of specific epitopes and antigen presenting cells for the treatment of many pathogenic infections and cancer. This paper provides a brief critical review of the current state of plasmid vaccines formulation and molecular delivery with analysis of performance data obtained from clinical trials.
    Matched MeSH terms: Plasmids
  12. Fulltext Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems
    Chan WT, Espinosa M, Yeo CC
    Front Mol Biosci, 2016;3:9.
    PMID: 27047942 DOI: 10.3389/fmolb.2016.00009
    In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I-VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains.
    Matched MeSH terms: Plasmids
  13. Survey of plasmids and resistance factors among veterinary isolates of Salmonella enteritidis in Malaysia
    Son R, Ansary A, Salmah I, Maznah A
    World J Microbiol Biotechnol, 1995 May;11(3):315-8.
    PMID: 24414656 DOI: 10.1007/BF00367107
    Thirty-five veterinary isolates of Salmonella enteritidis were characterized by their susceptibility to 10 antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All were susceptible to carbenicillin, chloramphenicol and nalidixic acid but 89% were resistant to tetracycline. When examined, 91% of the isolates harboured plasmids, with sizes ranging from 9.8 to 60 MDa. However, it was only possible to associate the presence of plasmids with tetracycline resistance; plasmids occurring in 90% of the tetracycline-resistant isolates. In conjugation experiments, with Escherichia coli K12 Nal(r) as recipient, the tetracycline resistance in three selected S. enteritidis isolates was observed to transfer at frequencies of 3.0×10(-3) to 1.0×10(-2)/donor cell. The concomitant transfer of a 56-MDa or 60-MDa plasmid in these three S. enteritidis isolates was also detected.
    Matched MeSH terms: Plasmids
  14. Fulltext First genomic insights into carbapenem-resistant Klebsiella pneumoniae from Malaysia
    Gan HM, Eng WWH, Dhanoa A
    J Glob Antimicrob Resist, 2020 03;20:153-159.
    PMID: 31325618 DOI: 10.1016/j.jgar.2019.07.008
    OBJECTIVES: Despite the increasing reports of carbapenem-resistant Enterobacteriaceae in Malaysia, genomic resources for carbapenem-resistant clinical strains of Klebsiella pneumoniae (K. pneumoniae) remain unavailable. This study aimed to sequence the genomes of multiple carbapenem-resistant K. pneumoniae strains from Malaysia and to identify the genetic basis for their resistance.

    METHODS: Illumina whole genome sequencing was performed on eight carbapenem-resistant K. pneumoniae isolated from a Malaysian hospital. Genetic diversity was inferred from the assembled genomes based on in silico multilocus sequence typing (MLST). In addition, plasmid-derived and chromosome-derived contigs were predicted using the machine learning approach. After genome annotation, genes associated with carbapenem resistance were identified based on similarity searched against the ResFinder database.

    RESULTS: The eight K. pneumoniae isolates were grouped into six different sequence types, some of which were represented by a single isolate in the MLST database. Genomic potential for carbapenem-resistance was attributed to the presence of plasmid-localised blaNDM (blaNDM-1/blaNDM-5) or blaKPC (blaKPC-2/blaKPC-6) in these sequenced strains. The majority of these carbapenem resistance genes was flanked by repetitive (transposase or integrase) sequences, suggesting their potential mobility. This study also reported the first blaKPC-6-harbouring plasmid contig to be assembled for K. pneumoniae, and the second for the genus Klebsiella.

    CONCLUSION: This study reported the first genomic resources for carbapenem-resistant K. pneumoniae from Malaysia. The high diversity of carbapenem resistance genes and sequence types uncovered from eight isolates from the same hospital is worrying and indicates an urgent need to improve the genomic surveillance of clinical K. pneumoniae in Malaysia.

    Matched MeSH terms: Plasmids
  15. Molecular characterization of Pasteurella multocida isolates from rabbits
    Al-Haddawi MH, Jasni S, Son R, Mutalib AR, Bahaman AR, Zamri-Saad M, et al.
    J Gen Appl Microbiol, 1999 Dec;45(6):269-275.
    PMID: 12501355
    Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.
    Matched MeSH terms: Plasmids
  16. Fulltext Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa
    Wong CF, Rahman RNZRA, Basri M, Salleh AB
    Iran J Biotechnol, 2017;15(3):194-200.
    PMID: 29845069 DOI: 10.15171/ijb.1524
    Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.
    Matched MeSH terms: Plasmids
  17. Influence of iron, growth temperature and plasmids on siderophore production in Aeromonas hydrophila
    Naidu AJ, Yadav M
    J Med Microbiol, 1997 Oct;46(10):833-8.
    PMID: 9364139
    Aeromonas hydrophila strains obtained from diarrhoeal samples of human patients (19 isolates) and freshwater ponds (11 isolates) were analysed for siderophore production. Both clinical and environmental isolates showed significantly increased siderophore production under iron-limiting conditions both at 28 degrees C and at 37 degrees C. Clinical isolates consistently produced higher levels of siderophores than did the environmental isolates. The role of plasmids in moderating siderophore production was studied after curing with acridine orange. Treatment with acridine orange for 24 h removed the larger plasmids but the smaller plasmids (< 5 MDa), more common in the environmental isolates, were resistant to curing. As found in the untreated isolates, the cured clinical isolates produced higher mean levels of siderophores than the cured environmental isolates. Siderophore production in A. hydrophila was significantly influenced by iron-limiting cultural conditions and the source of isolates, but plasmid content and growth temperature at 28 degrees C or 37 degrees C had little effect on production. The basis for the greater production of siderophores in clinical isolates than in environmental isolates needs further study.
    Matched MeSH terms: Plasmids
  18. Fulltext Cloning of gene encoding yeast alcohol dehydrogenase 1 (YADH 1) in Escherichia coli TOP10 for biocatalysis
    Mohd Rezuan M Aspar, Rashidah Abdul Rahim, Mohamad Hekarl Uzir
    Pertanika Journal of Science & Technology, 2011;19(2):423-430.
    MyJurnal
    Yeast producing alcohol dehydrogenase 1 (YADH 1) enzyme has been used as a biocatalyst for the synthesis of an optically active flavouring compound known as citronellol. However, the slow growth of yeast (Saccharomyces cerevisiae) has deterred the progress of biotransformation. The main purpose of this work is to clone the genes producing YADH1 enzyme from yeast into a faster growing bacteria, Escherichia coli. Initially, the sequence of the gene encoding this protein has been identified in the S. cerevisiae Genome Databases (SGD). The so-called Yadh1 gene sequence is located from coordinate 159548 to 160594 on chromosome XV of yeast. Based on this information, two primer sequences (Forward and Reverse) were constructed. Each of these primers will bind to either end of the Yadh1 gene. The Yadh1 gene was then amplified using Polymerase Chain Reaction (PCR) technique. The amplified Yadh 1 gene was successfully cloned into a cloning vector, TOPO TA plasmid. This plasmid also contains a gene which confers resistance to ampicillin. This recombinant
    plasmid was then inserted into Escherichia coli TOP 10 using heat shock protocol at 42oC. Finally, the cloned bacteria containing the recombinant TOPO TA plasmid harbouring Yadh1 gene was able to grow on Luria Bertani (LB) media supplied with antibiotic.
    Matched MeSH terms: Plasmids
  19. Large BACs transfect more efficiently in circular topology
    Wong YC, Osahor A, Al-Ajli FOM, Narayanan K
    Anal Biochem, 2021 10 01;630:114324.
    PMID: 34363787 DOI: 10.1016/j.ab.2021.114324
    The effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells.
    Matched MeSH terms: Plasmids
  20. Fulltext The complete genome sequence of Ensifer meliloti strain CCMM B554 (FSM-MA), a highly effective nitrogen-fixing microsymbiont of Medicago truncatula Gaertn
    Nagymihály M, Vásarhelyi BM, Barrière Q, Chong TM, Bálint B, Bihari P, et al.
    Stand Genomic Sci, 2017;12:75.
    PMID: 29255570 DOI: 10.1186/s40793-017-0298-3
    Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA.
    Matched MeSH terms: Plasmids
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