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Fulltext Screening for New Delhi metallo-β-lactamase-1 in Enterobacteriaceae: Is there a role for the modified Hodge test?

Abidin NZ, Sulong A, Alfizah H, Ding CH, Muttaqillah NA, Rahman MM

Pak J Med Sci, 2015 Nov-Dec;31(6):1340-3.
PMID: 26870093 DOI: 10.12669/pjms.316.8159

The New Delhi metallo-β-lactamase-1 (NDM-1) enzyme is a plasmid-encoded enzyme that inactivates carbapenem antibiotics. This study aims to ascertain if the modified Hodge test (MHT) has a role in screening for NDM-1 in Enterobacteriaceae with reduced carbapenem susceptibility.

Matched MeSH terms: Plasmids
Characterization and Comparative Overview of Complete Sequences of the First Plasmids of Pandoraea across Clinical and Non-clinical Strains

Yong D, Tee KK, Yin WF, Chan KG

Front Microbiol, 2016;7:1606.
PMID: 27790203

To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572(T) (pPF72-1, pPF72-2), Pandoraea oxalativorans DSM 23570(T) (pPO70-1, pPO70-2, pPO70-3, pPO70-4), Pandoraea vervacti NS15 (pPV15) and Pandoraea apista DSM 16535(T) (pPA35) were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful as the sequences did not match any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA, and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified amongst the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens.

Matched MeSH terms: Plasmids
Plasmid localization of sole rrn operon in genomes of Oecophyllibacter saccharovorans (Acetobacteraceae)

Chua KO, See-Too WS, Yong HS, Song SL, Yin WF, Chan KG

Plasmid, 2021 03;114:102559.
PMID: 33476637 DOI: 10.1016/j.plasmid.2021.102559

The bacterium Oecophyllibacter saccharovorans of family Acetobacteraceae is a symbiont of weaver ant Oecophylla smaragdina. In our previous study, we published the finding of novel O. saccharovorans strains Ha5T, Ta1 and Jb2 (Chua et al. 2020) but their plasmid sequences have not been reported before. Here, we demonstrate for the first time that the sole rrn operon of their genomes was detected on a 6.6 kb circular replicon. This replicon occurred in high copy number, much smaller size and lower G + C content than the main chromosome. Based on these features, the 6.6 kb circular replicon was regarded as rrn operon-containing plasmid. Further restriction analysis on the plasmids confirmed their circular conformation. A Southern hybridization analysis also corroborated the presence of 16S rRNA gene and thus the rrn operon on a single locus in the genome of the O. saccharovorans strains. However, similar genome architecture was not observed in other closely related bacterial strains. Additional survey also detected no plasmid-borne rrn operon in available genomes of validly described taxa of family Acetobacteraceae. To date, plasmid localization of rrn operon is rarely documented. This study reports the occurrence of rrn operon on the smallest bacterial plasmid in three O. saccharovorans strains and discusses its possible importance in enhancing their competitive fitness as bacterial symbiont of O. smaragdina.

Matched MeSH terms: Plasmids/genetics
Analysis of CRISPR-Cas Loci and their Targets in Levilactobacillus brevis

Goh YX, Wang M, Hou XP, He Y, Ou HY

Interdiscip Sci, 2023 Sep;15(3):349-359.
PMID: 36849628 DOI: 10.1007/s12539-023-00555-1

The CRISPR‒Cas system acts as a bacterial defense mechanism by conferring adaptive immunity and limiting genetic reshuffling. However, under adverse environmental hazards, bacteria can employ their CRISPR‒Cas system to exchange genes that are vital for adaptation and survival. Levilactobacillus brevis is a lactic acid bacterium with great potential for commercial purposes because it can be genetically manipulated to enhance its functionality and nutritional value. Nevertheless, the CRISPR‒Cas system might interfere with the genetic modification process. Additionally, little is known about the CRISPR‒Cas system in this industrially important microorganism. Here, we investigate the prevalence, diversity, and targets of CRISPR‒Cas systems in the genus Levilactobacillus, further focusing on complete genomes of L. brevis. Using the CRISPRCasFinder webserver, we identified 801 putative CRISPR-Cas systems in the genus Levilactobacillus. Further investigation focusing on the complete genomes of L. brevis revealed 54 putative CRISPR-Cas systems. Of these, 46 were orphan CRISPRs, and eight were CRISPR‒Cas systems. The type II-A CRISPR‒Cas system is the most common in Levilactobacillus and L. brevis complete genomes. Analysis of the spacer's target showed that the CRISPR‒Cas systems of L. brevis mainly target the enterococcal plasmids. Comparative analysis of putative CRISPR-Cas loci in Levilactobacillus brevis.

Matched MeSH terms: Plasmids/genetics
Fulltext Plasmids in penicillinase-producing Neisseria gonorrhoeae in Peninsular Malaysia

Koh CL, Kalaimathee KK, Ngeow YF

Med J Malaysia, 1984 Dec;39(4):269-71.
PMID: 6443581

The plasmid profiles of 66 strains of penicillinase-producing Neisseria gonorrhoeae (PPNG), isolated in Peninsular Malaysia, were determined by agarose gel electrophoresis. All the isolates harboured two common plasmid species, a 4.4 x 10 6 Far-East type R plasmid associated with beta-lactamase production and a 2.6 x 106 plasmid of unknown function. In addition to these two plasmids, 51 (77%) PPNG isolates also carried a 24.5 x 106 conjugative plasmid.

Matched MeSH terms: Plasmids*
Fulltext Characterization of multidrug-resistant Acinetobacter baumannii strain ATCC BAA1605 using whole-genome sequencing

Ten KE, Md Zoqratt MZH, Ayub Q, Tan HS

BMC Res Notes, 2021 Mar 04;14(1):83.
PMID: 33663564 DOI: 10.1186/s13104-021-05493-z

OBJECTIVE: The nosocomial pathogen, Acinetobacter baumannii, has acquired clinical significance due to its ability to persist in hospital settings and survive antibiotic treatment, which eventually resulted in the rapid spread of this bacterium with antimicrobial resistance (AMR) phenotypes. This study used a multidrug-resistant A. baumannii (strain ATCC BAA1605) as a model to study the genomic features of this pathogen.
RESULTS: One circular chromosome and one circular plasmid were discovered in the complete genome of A. baumannii ATCC BAA1605 using whole-genome sequencing. The chromosome is 4,039,171 bp long with a GC content of 39.24%. Many AMR genes, which confer resistance to major classes of antibiotics (beta-lactams, aminoglycosides, tetracycline, sulphonamides), were found on the chromosome. Two genomic islands were predicted on the chromosome, one of which (Genomic Island 1) contains a cluster of AMR genes and mobile elements, suggesting the possibility of horizontal gene transfer. A subtype I-F CRISPR-Cas system was also identified on the chromosome of A. baumannii ATCC BAA1605. This study provides valuable genome data that can be used as a reference for future studies on A. baumannii. The genome of A. baumannii ATCC BAA1605 has been deposited at GenBank under accession no. CP058625 and CP058626.

Matched MeSH terms: Plasmids/genetics
An improved direct metamobilome approach increases the detection of larger-sized circular elements across kingdoms

Alanin KWS, Jørgensen TS, Browne PD, Petersen B, Riber L, Kot W, et al.

Plasmid, 2021 05;115:102576.
PMID: 33872684 DOI: 10.1016/j.plasmid.2021.102576

Mobile genetic elements (MGEs) are instrumental in natural prokaryotic genome editing, permitting genome plasticity and allowing microbes to accumulate genetic diversity. MGEs serve as a vast communal gene pool and include DNA elements such as plasmids and bacteriophages (phages) among others. These mobile DNA elements represent a human health risk as they can introduce new traits, such as antibiotic resistance or virulence, to a bacterial strain. Sequencing libraries targeting environmental circular MGEs, referred to as metamobilomes, may broaden our current understanding of the mechanisms behind the mobility, prevalence and content of these elements. However, metamobilomics is affected by a severe bias towards small circular elements, introduced by multiple displacement amplification (MDA). MDA is typically used to overcome limiting DNA quantities after the removal of non-circular DNA during library preparations. By examining the relationship between sequencing coverage and the size of circular MGEs in paired metamobilome datasets with and without MDA, we show that larger circular elements are lost when using MDA. This study is the first to systematically demonstrate that MDA is detrimental to detecting larger-sized plasmids if small plasmids are present. It is also the first to show that MDA can be omitted when using enzyme-based DNA fragmentation and PCR in library preparation kits such as Nextera XT® from Illumina.

Matched MeSH terms: Plasmids/genetics
Electrotransformation of thermophilic bacterium Caldimonas manganoxidans

Arai T, Aikawa S, Sudesh K, Kondo T, Kosugi A

J Microbiol Methods, 2022 01;192:106375.
PMID: 34793853 DOI: 10.1016/j.mimet.2021.106375

Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/μg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.

Matched MeSH terms: Plasmids/genetics
Construction of molecule markers for prolific oil palm tissue culture

Siti Khadijah A. Karim, Nik Marzuki Sidik

Sains Malaysiana, 2018;47:1701-1708.

Penggunaan penanda DNA boleh mengurangkan masalah dalam kultur tisu khususnya apabila diaplikasikan semasa
pemilihan pokok untuk kultur tisu. Oleh itu, penyelidikan ini dijalankan bertujuan untuk membina penanda molekul
bagi kultur tisu kelapa sawit prolifik dengan menggunakan teknik polimorfisme panjang cebisan teramplifikasi (AFLP).
Analisis AFLP dijalankan ke atas 20 klon kelapa sawit yang terbahagi kepada tiga kelas iaitu klon tidak prolifik (10
jenis klon), klon normal (6 jenis klon) dan klon prolifik (4 jenis klon). Kesemua klon yang digunakan adalah daripada
titisan sel yang berbeza. Sebanyak 25 kombinasi pencetus telah digunakan dalam analisis AFLP dan 13 daripada mereka
memberikan corak amplifikasi polimorfisme. Daripada hasil ini, sebanyak 44 cebisan polimorfik telah dipencilkan dengan
33 cebisan adalah bagi klon tidak prolifik, 1 cebisan bagi normal dan 10 cebisan bagi klon prolifik. Cebisan ini telah
diklon ke dalam plasmid, berjujukan dan seterusnya, analisis jujukan dijalankan. Sebanyak 36 cebisan polimorfik telah
digunakan bagi kajian seterusnya. Berdasarkan kepada jujukan yang diperoleh, sepasang pencetus yang khusus kepada
setiap cebisan telah dijana. Jangkaan julat saiz jalur DNA yang diamplifikasi bagi setiap pencetus adalah antara 70
hingga 500 bp. Pasangan pencetus yang optimum diuji ke atas 20 jenis klon kelapa sawit untuk mengesahkan penanda
yang telah dibina. Daripada 36 pasangan pencetus yang dibina, 2 pasang pencetus telah menunjukkan potensi untuk
digunakan sebagai penanda kepada kultur tisu kelapa sawit prolifik.

Matched MeSH terms: Plasmids
Fulltext Diagnosis and potential treatments for acute hepatopancreatic necrosis disease (AHPND): a review

Santos HM, Tsai CY, Maquiling KRA, Tayo LL, Mariatulqabtiah AR, Lee CW, et al.

Aquac Int, 2020;28(1):169-185.
PMID: 32834683 DOI: 10.1007/s10499-019-00451-w

Acute hepatopancreatic necrosis disease (AHPND) or formerly known as early mortality syndrome (EMS) is an emerging disease that has caused significant economic losses to the aquaculture industry. The primary causative agent of AHPND is Vibrio parahaemolyticus, a Gram-negative rod-shaped bacterium that has gained plasmids encoding the fatal binary toxins Pir A/Pir B that cause rapid death of the infected shrimp. In this review, the current research studies and information about AHPND in shrimps have been presented. Molecular diagnostic tools and potential treatments regarding AHPND were also included. This review also includes relevant findings which may serve as guidelines that can help for further investigation and studies on AHPND or other shrimp diseases.

Matched MeSH terms: Plasmids
Fulltext Rapid Generation of a Recombinant Genotype VIII Newcastle Disease Virus (NDV) Using Full-Length Synthetic cDNA

Murulitharan K, Yusoff K, Omar AR, Peeters BPH, Molouki A

Curr Microbiol, 2021 Apr;78(4):1458-1465.
PMID: 33660046 DOI: 10.1007/s00284-021-02421-z

Rescue of (-)ssRNA viruses involves the sequential assembly and cloning of the full-length cDNA, which is often a challenging and time-consuming process. The objective of this study was to develop a novel method to rapidly clone the full-length cDNA of a very virulent NDV by only one assembly step. A completely synthetic 15 kb cDNA of a Malaysian genotype VIII NDV known as strain AF2240-I with additional flanking BsmBI sites was synthesised. However, to completely follow the rule-of-six, the additional G residues that are traditionally added after the T7 promoter transcription initiation site were not synthesised. The synthetic fragment was then cloned into low-copy number transcription vector pOLTV5-phiX between the T7 promoter and HDV Rz sequences through digestion with BbsI. The construct was co-transfected with helper plasmids into BSRT7/5 cells. A recombinant NDV called rAF was successfully rescued using transfection supernatant harvested as early as 16 h post-transfection. Virus from each passage showed an intracerebral pathogenicity index (ICPI) and a mean death time (MDT) similar to the parent strain AF2240-I. Moreover, rAF possessed an introduced mutation which was maintained for several passages. The entire rescue using the one-step assembly procedure was completed within a few weeks, which is extremely fast compared to previously used methods.

Matched MeSH terms: Plasmids
Sistem minigenom virus Penyakit Newcastle (NDV) AF2240 sebagai sistem pengekspresan gen sel mamalia

SHAHRUL HISHAM ZAINAL ARIFFIN, ROSLINA SHAMSUDIN, NURUL ATIKAH AHMAD, ZULKIFLIE ZAMROD

Sains Malaysiana, 2012;41:423-430.

Sistem minigenom telah digunakan untuk mengkaji replikasi dan transkripsi virus RNA tidak bersegmen. Objektif kajian ini adalah untuk membina sistem minigenom bagi virus NDV strain tempatan, AF2240 serta bagi mengkaji mekanisme transkripsi dan replikasi virus ini. Bagi tujuan ini lima plasmid digunakan iaitu pMGNDV, pCITENP, pCITEP, pTriEX-T7, dan pGEML. Kesemua plasmid diekstrak secara berskala besar dan dimendakkan menggunakan polietilina glikol. Hasil ekstrak ini digunakan untuk transfeksi ke dalam sel. Translasi in vitro dilakukan dengan menggunakan pCITENP, pCITEP, dan pTriEX-T7 untuk memastikan kesemua konstruk ini berfungsi. Hasil pemblotan western menunjukkan protein bersaiz ~100 kDa (T7), ~53 kDa (NP), ~53 dan 55 kDa (P) berjaya diekspreskan. Protein CAT diperoleh apabila plasmid yang mengekodkan minigenom NDV ditransfeksi bersama plasmid yang mengekodkan protein nukleokapsid (NP), fosfoprotein (P) dan subunit besar polimerase (L) ke dalam sel BHK-21. Dianggarkan 55 pg protein CAT berjaya diperoleh menggunakan kit CAT ELISA. Hasil pemblotan western turut menunjukkan protein CAT bersaiz 25 kDa dihasilkan. Kesimpulannnya, system minigenom ini berupaya untuk berfungsi dan mampu mengekspreskan gen asing di dalam sel mamalia BHK-21.

Matched MeSH terms: Plasmids
Fulltext Increased recovery and improved purity of PHA from recombinant Cupriavidus necator

Anis SN, Iqbal NM, Kumar S, Al-Ashraf A

Bioengineered, 2013 Mar-Apr;4(2):115-8.
PMID: 23018620 DOI: 10.4161/bioe.22350

A simple procedure for recovering biodegradable polymer from bacterial cells has been developed using economical and environmentally friendly solvent or chemicals. Recombinant bacterium, Cupriavidus necator harboring pBBR1MCS-C2 plasmid polyhydroxyalkanoate (PHA) synthase gene was used for the production of copolymer P(3HB-co-3HHx) from crude palm kernel oil (CPKO). NaOH was chosen in this study as it could give high purity and recovery yield. Increase of NaOH concentration had resulted in an increase of the PHA purity, but the recovery yield had decreased. The greater improvement of PHA purity and recovery were achieved by incubating the freeze-dried cells (10-30 g/L) in NaOH (0.1 M) for 1-3 h at 30°C and polishing using 20% (v/v) of ethanol. The treatment caused negligible degradation of the molecular weight of PHA recovered from the bacterial cells. The present review also highlights other extraction methods to provide greater insights into economical and sustainable recovery of PHA from bacterial cells.

Matched MeSH terms: Plasmids/genetics
Plasmid incidence rate and conjugative chloramphenicol and tetracycline resistance plasmids in Malaysian isolates of Salmonella typhi

Phipps M, Pang T, Koh CL, Puthucheary S

Microbiol. Immunol., 1991;35(2):157-61.
PMID: 1886492

Seven (6.1%) of 115 strains of Salmonella typhi isolated from Malaysian patients harbored a single large plasmid of 71 to 166 mD. Two of the seven plasmid-bearing strains were resistant to chloramphenicol (Cm) and tetracycline (Tc) and they transferred Cm and Tc resistance traits to Escherichia coli K12 at frequencies from 1.6 x 10(-7) to 1.9 x 10(-6). Agarose gel electrophoresis provided evidence that the resistance traits were cotransferred on a conjugative plasmid. The significance and importance of these results are discussed.

Matched MeSH terms: Plasmids*
Complete genome of Jeotgalibacillus malaysiensis D5(T) consisting of a chromosome and a circular megaplasmid

Goh KM, Chan KG, Yaakop AS, Ee R

J Biotechnol, 2015 Jun 20;204:13-4.
PMID: 25858153 DOI: 10.1016/j.jbiotec.2015.03.007

Jeotgalibacillus spp. are halophilic bacteria within the family Planococcaceae. No genomes of Jeotgalibacillus spp. have been reported to date, and their metabolic pathways are unknown. How the bacteria survive in hypertonic conditions such as seawater is yet to be discovered. As only few studies have been conducted on Jeotgalibacillus spp., potential applications of these bacteria are unknown. Here, we present the complete genome of J. malaysiensis D5(T) (=DSM 28777(T) =KCTC 33350(T)), which is invaluable in identifying interesting applications for this genus.

Matched MeSH terms: Plasmids/genetics
Fulltext Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Hassan MI, McSorley FR, Hotta K, Boddy CN

J Vis Exp, 2017 06 27.
PMID: 28715370 DOI: 10.3791/55187

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

Matched MeSH terms: Plasmids/genetics*
Fulltext Comparative genomic analysis of clinical Acinetobacter nosocomialis isolates from Terengganu, Malaysia led to the discovery of a novel tetracycline-resistant plasmid

Mohd Rani F, Lean SS, A Rahman NI, Ismail S, Alattraqchi AG, Amonov M, et al.

J Glob Antimicrob Resist, 2022 Dec;31:104-109.
PMID: 36049733 DOI: 10.1016/j.jgar.2022.08.019

OBJECTIVES: To analyse the genome sequences of four archival Acinetobacter nosocomialis clinical isolates (designated AC13, AC15, AC21 and AC25) obtained from Terengganu, Malaysia in 2011 to determine their genetic relatedness and basis of antimicrobial resistance.
METHODS: Antimicrobial susceptibility profiles of the A. nosocomialis isolates were determined by disk diffusion. Genome sequencing was performed using the Illumina NextSeq platform.
RESULTS: The four A. nosocomialis isolates were cefotaxime resistant whereas three isolates (namely, AC13, AC15 and AC25) were tetracycline resistant. The carriage of the blaADC-255-encoded cephalosporinase gene is likely responsible for cefotaxime resistance in all four isolates. Phylogenetic analysis indicated that the three tetracycline-resistant isolates were closely related, with an average nucleotide identity of 99.9%, suggestive of nosocomial spread, whereas AC21 had an average nucleotide identity of 97.9% when compared to these three isolates. The tetracycline-resistant isolates harboured two plasmids: a 13476 bp Rep3-family plasmid of the GR17 group designated pAC13-1, which encodes the tetA(39) tetracycline-resistance gene, and pAC13-2, a 4872 bp cryptic PriCT-1-family plasmid of a new Acinetobacter plasmid group, GR60. The tetA(39) gene was in a 2 001 bp fragment flanked by XerC/XerD recombination sites characteristic of a mobile pdif module. Both plasmids also harboured mobilisation/transfer-related genes.
CONCLUSIONS: Genome sequencing of A. nosocomialis isolates led to the discovery of two novel plasmids, one of which encodes the tetA(39) tetracycline-resistant gene in a mobile pdif module. The high degree of genetic relatedness among the three tetracycline-resistant A. nosocomialis isolates is indicative of nosocomial transmission.

Matched MeSH terms: Plasmids/genetics
Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells

Wong YC, Ng AWR, Chen Q, Liew PS, Lee CW, Sim EUH, et al.

ACS Synth Biol, 2023 Apr 21;12(4):909-921.
PMID: 37026178 DOI: 10.1021/acssynbio.2c00580

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

Matched MeSH terms: Plasmids/genetics
Cellulose acetate phthalate microencapsulation and delivery of plasmid DNA to the intestines

Hanafi A, Nograles N, Abdullah S, Shamsudin MN, Rosli R

J Pharm Sci, 2013 Feb;102(2):617-26.
PMID: 23192729 DOI: 10.1002/jps.23389

Cellulose acetate phthalate (CAP) microcapsules were formulated to deliver plasmid DNA (pDNA) to the intestines. The microcapsules were characterized and were found to have an average diameter of 44.33 ± 30.22 μm, and were observed to be spherical with smooth surface. The method to extract pDNA from CAP was modified to study the release profile of the pDNA. The encapsulated pDNA was found to be stable. Exposure to the acidic and basic pH conditions, which simulates the pH environment in the stomach and the intestines, showed that the release occurred in a stable manner in the former, whereas it was robust in the latter. The loading capacity and encapsulation efficiency of the microcapsules were low but the CAP recovery yield was high which indicates that the microcapsules were efficiently formed but the loading of pDNA can be improved. In vitro transfection study in 293FT cells showed that there was a significant percentage of green-fluorescent-protein-positive cells as a result of efficient transfection from CAP-encapsulated pDNA. Biodistribution studies in BALB/c mice indicate that DNA was released at the stomach and intestinal regions. CAP microcapsules loaded with pDNA, as described in this study, may be useful for potential gene delivery to the intestines for prophylactic or therapeutic measures for gastrointestinal diseases.

Matched MeSH terms: Plasmids/administration & dosage; Plasmids/chemical synthesis*; Plasmids/genetics
Purification of transcriptionally active multimeric plasmid DNA using zwitterionic detergent and carbonate apatite nano-particles

Tee LK, Ling CS, Chua MJ, Abdullah S, Rosli R, Chowdhury EH

Plasmid, 2011 Oct;66(1):38-46.
PMID: 21419794 DOI: 10.1016/j.plasmid.2011.03.001

Plasmid DNA is one of the indispensable components in molecular biology research and a potential biomaterial for gene therapy and DNA vaccination. Both quality and quantity of extracted plasmid DNA are of the great interests in cloning and subsequent expression of genes in vitro and in vivo for basic research and therapeutic interventions. Bacteria with extremely short generation times are the valuable source of plasmid DNA that can be isolated through a number of existing techniques. However, the current methods have some limitations in isolating high quality plasmid DNA since the multimeric plasmid which is believed to be more efficiently transcribed by RNA polymerase than the monomeric form, is almost lost during the extraction process. Recently, we developed a rapid isolation technique for multimeric plasmid based on generation of a 'protein aggregate' using a zwitterionic detergent and alkali. Here we have investigated the roles of different parameters in the whole extraction process to optimise the production of high quality multimeric plasmid DNA. Moreover, we have showed the advantageous effects of nanoparticles to effectively sediment the 'protein aggregate' for smooth elution of multimeric plasmid DNA from it. Finally, quality assessment study has revealed that the isolated multimeric DNA is at least 10 times more transcriptionally active than the monomeric form isolated by the commercially available Qiaget kit.

Matched MeSH terms: Plasmids/genetics; Plasmids/isolation & purification*; Plasmids/chemistry

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