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Plasmid curing

Ida Lauritsen, Se Hyeuk Kim, Andreas Porse, Morten H H Nørholm
Plasmids are highly useful tools for studying living cells and for heterologous expression of genes and pathways in cell factories. Standardized tools and operating procedures for handling such DNA vectors are core principles in synthetic biology. Here, we describe protocols for molecular cloning and exchange of genetic parts in the Standard European Vectors Architecture (SEVA) vector system. Additionally, to facilitate rapid testing and iterative bioengineering using different vector designs, we provide a one-step protocol for a universal CRISPR-Cas9-based plasmid curing system (pFREE) and demonstrate the application of this system to cure SEVA constructs (all vectors are available at SEVA/Addgene)...
2018: Methods in Molecular Biology
Kyeong Rok Choi, Jae Sung Cho, In Jin Cho, Dahyeon Park, Sang Yup Lee
Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida, an essential strategy to develop stable industrial strains producing heterologous bioproducts, requires development of a more efficient method. Current methods rely on time-consuming homologous recombination techniques and transposon-mediated random insertions...
May 8, 2018: Metabolic Engineering
Laura Fernández, Eugenia Carrillo, Lucas Sánchez-Sampedro, Carmen Sánchez, Ana Victoria Ibarra-Meneses, M Angeles Jimenez, Valter Dos Anjos Almeida, Mariano Esteban, Javier Moreno
Leishmania -activated C-kinase antigen (LACK) is a highly conserved protein among Leishmania species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65) expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL). Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas...
2018: Frontiers in Immunology
Tobias Schilling, Sascha Dietrich, Michael Hoppert, Robert Hertel
Phages are currently under discussion as a solution for the antibiotic crisis, as they may cure diseases caused by multi-drug-resistant pathogens. However, knowledge of phage biology and genetics is limited, which impedes risk assessment of therapeutic applications. In order to enable advances in phage genetic research, the aim of this work was to create a toolkit for simple and fast genetic engineering of phages recruiting Bacillus subtilis as host system. The model organism B. subtilis represents a non-pathogenic surrogate of its harmful relatives, such as Bacillus anthracis or Bacillus cereus ...
May 4, 2018: Viruses
Chao Yin, Lijuan Xu, Yang Li, Zijian Liu, Dan Gu, Qiuchun Li, Xinan Jiao
In Salmonella, plasmids participate in many pathways involved in virulence, metabolism, and antibiotic resistance. To investigate the function of the ipaJ gene in a multi-copy plasmid pSPI12 prevalent in Salmonella enterica serovar Pullorum (S. Pullorum), we established a method to eliminate plasmid and constructed the plasmid-cured bacteria C79-13-ΔpSPI12 by using the suicide vector pDM4. Briefly, a 500 bp fragment ipaJU from pSPI12 was cloned into pDM4 and transformed into S. Pullorum C79-13 by conjugative transfer...
May 1, 2018: Avian Pathology: Journal of the W.V.P.A
Evangelia S Papadopoulou, Chiara Perruchon, Sotirios Vasileiadis, Constantina Rousidou, Georgia Tanou, Martina Samiotaki, Athanassios Molassiotis, Dimitrios G Karpouzas
Diphenylamine (DPA) is a common soil and water contaminant. A Pseudomonas putida strain, recently isolated from a wastewater disposal site, was efficient in degrading DPA. Thorough knowledge of the metabolic capacity, genetic stability and physiology of bacteria during biodegradation of pollutants is essential for their future industrial exploitation. We employed genomic, proteomic, transcription analyses and plasmid curing to (i) identify the genetic network of P. putida driving the microbial transformation of DPA and explore its evolution and origin and (ii) investigate the physiological response of bacterial cells during degradation of DPA...
2018: Frontiers in Microbiology
Andrew D Huber, Jennifer J Wolf, Dandan Liu, Anna T Gres, Jing Tang, Kelsey N Boschert, Maritza N Puray-Chavez, Dallas L Pineda, Thomas G Laughlin, Emily M Coonrod, Qiongying Yang, Juan Ji, Karen A Kirby, Zhengqiang Wang, Stefan G Sarafianos
Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly in vitro have been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp in vitro and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs...
April 25, 2018: MSphere
Rachel J Skilton, Yibing Wang, Colette O'Neill, Simone Filardo, Peter Marsh, Angèle Bénard, Nicholas R Thomson, Kyle H Ramsey, Ian N Clarke
Background: Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence.  Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle...
2018: Wellcome Open Research
Deniz Aksoy, Ece Şen
Poultry animals and poultry associated products are important risk sources for Salmonellosis. S.Kentucky and S.Infantis are among the serovars frequently isolated from retail chickens and were reported to be isolated in Turkey. In this study, the role of plasmids carried by S.Kentucky and S.Infantis isolates in antibiotic resistance profiles of the isolates and their pathogenicity on Caenorhabditis elegans nematode model system were investigated. The isolates used, 1 of Kentucky and 2 of Infantis serotypes, were selected among food-borne Salmonella isolated from chicken carcass in Edirne...
January 2018: Mikrobiyoloji Bülteni
Jana Minarovičová, Adriana Véghová, Mária Mikulášová, Romana Chovanová, Katarína Šoltýs, Hana Drahovská, Eva Kaclíková
Listeria monocytogenes is a serious foodborne pathogen capable of persisting in food processing environments. Tolerance to disinfectants used in industrial settings constitutes an important factor of Listeria survival. In the present study, the mechanism of tolerance to benzalkonium chloride (BAC) was investigated in 77 L. monocytogenes isolates from a meat facility. By PCR approach, the mdrL and lde chromosomal efflux pump genes were detected in all isolates. No isolate was positive for qacH and emrE genes...
April 6, 2018: Antonie Van Leeuwenhoek
Cuicui Lyu, Jun Shen, Rui Wang, Haihui Gu, Jianping Zhang, Feng Xue, Xiaofan Liu, Wei Liu, Rongfeng Fu, Liyan Zhang, Huiyuan Li, Xiaobing Zhang, Tao Cheng, Renchi Yang, Lei Zhang
BACKGROUND: Replacement therapy for hemophilia remains a lifelong treatment. Only gene therapy can cure hemophilia at a fundamental level. The clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9 (CRISPR-Cas9) system is a versatile and convenient genome editing tool which can be applied to gene therapy for hemophilia. METHODS: A patient's induced pluripotent stem cells (iPSCs) were generated from their peripheral blood mononuclear cells (PBMNCs) using episomal vectors...
April 6, 2018: Stem Cell Research & Therapy
Masafumi Noda, Masaya Shiraga, Takanori Kumagai, Narandalai Danshiitsoodol, Masanori Sugiyama
Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0 : 5.7 : 1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3...
2018: Biological & Pharmaceutical Bulletin
Suneeta Chimalapati, Marcela de Souza Santos, Kelly Servage, Nicole J De Nisco, Ankur B Dalia, Kim Orth
The Gram-negative bacterium Vibrio parahaemolyticus is an opportunistic human pathogen and the leading cause of seafood borne acute gastroenteritis worldwide. Recently, this bacterium was implicated as the etiologic agent of a severe shrimp disease with consequent devastating outcomes to shrimp farming. In both cases, acquisition of genetic material via horizontal transfer provided V. parahaemolyticus with new virulence tools to cause disease. Dissecting the molecular mechanisms of V. parahaemolyticus pathogenesis often requires manipulating its genome...
March 19, 2018: Journal of Bacteriology
M Giles, S A Cawthraw, M AbuOun, C M Thomas, D Munera, M K Waldor, R M La Ragione, J M Ritchie
Objectives: To assess stability and contribution of a large ESBL-encoding IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts. Methods: Specific-pathogen-free 3-4-day-old New Zealand White rabbits and conventionally reared 6-week-old weaned lambs were orally infected with WT E. coli O104:H4 or the ESBL-plasmid-cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time...
February 28, 2018: Journal of Antimicrobial Chemotherapy
Yu Liu, Miaoxian Zhao, Mingxing Gong, Ying Xu, Cantao Xie, Haohui Deng, Xueying Li, Hongkai Wu, Zhanhui Wang
Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector...
April 2018: Antiviral Research
Simon van der Els, Jennelle K James, Michiel Kleerebezem, Peter A Bron
CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci...
April 15, 2018: Applied and Environmental Microbiology
Karolina Pierzynowska, Lidia Gaffke, Aleksandra Hać, Jagoda Mantej, Natalia Niedziałek, Joanna Brokowska, Grzegorz Węgrzyn
Huntington's disease (HD) is a monogenic disorder, caused by mutations in the HTT gene which result in expansion of CAG triplets. The product of the mutated gene is misfolded huntingtin protein that forms aggregates leading to impairment of neuronal function, neurodegeneration, motor abnormalities and cognitive deficits. No effective cure is currently available for HD. Here we studied effects of genistein (trihydroxyisoflavone) on a HD cellular model consisting of HEK-293 cells transfected with a plasmid bearing mutated HTT gene...
March 2018: Neuromolecular Medicine
Xiaohong Liu, Chenglong Jiao, Yue Ma, Qiyao Wang, Yuanxing Zhang
Tiger puffer (Takifugu rubripes) is becoming an economic promising aquaculture species in China. However, the development of Tiger puffer breeding industry is restricted by some serious aquatic disease such as vibriosis. An effective live attenuated vaccine MVAV6203 was developed in our previous studies by curing the virulence plasmid pEIB1 and unmarked inframe-deletion of the aroC gene from the virulent V. anguillarum. Here, we evaluated whether this live vaccine was suitable for Tiger puffer against disease caused by Vibrio genus...
March 7, 2018: Vaccine
Xin Song, He Huang, Zhiqiang Xiong, Yongjun Xia, Guangqiang Wang, Boxing Yin, Lianzhong Ai
The cryptic plasmid pLC2W was isolated from Lactobacillus casei CP002616. Nucleotide sequence analysis revealed that 4 putative open reading frames (ORF) were responsible for DNA replication. Four Escherichia coli-Lactobacillus shuttle vectors were constructed using different lengths of the pLC2W replicon to identify the shortest functional replicon. The length of the pLC2W replicon did not affect the stability of the plasmids. Green fluorescent protein (GFP) as a reporter was expressed successfully in several lactobacilli using our constructed vectors...
April 2018: Journal of Dairy Science
Michael John Patton, Chih-Yu Chen, Chunfu Yang, Stuart McCorrister, Chris Grant, Garrett Westmacott, Xin-Yong Yuan, Estela Ochoa, Robert Fariss, William M Whitmire, John H Carlson, Harlan D Caldwell, Grant McClarty
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachoma-which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections...
January 30, 2018: MBio
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