Methods for the genetic manipulation of marine bacteria

Genetic manipulation of bacteria is a procedure necessary to obtain new strains that express peculiar and defined genetic determinants or to introduce genetic variants responsible for phenotypic modifications. This procedure can be applied to explore the biotechnological potential associated with environmental bacteria and to utilize the functional properties of specific genes when inserted into an appropriate host. In the past years, marine bacteria have received increasing attention because they represent a fascinating reservoir of genetic and functional diversity that can be utilized to fuel the bioeconomy sector. However, there is an urgent need for an in-depth investigation and improvement of the genetic manipulation tools applicable to marine strains because of the paucity of knowledge regarding this. This review aims to describe the genetic manipulation methods hitherto used in marine bacteria, thus highlighting the limiting factors of the different techniques available today to increase manipulation efficiency. In particular, we focus on methods of natural and artificial transformations (especially electroporation) and conjugation because they have been successfully applied to several marine strains. Finally, we emphasize that, to avoid failure, future work should be carried out to establish tailored methodologies for marine bacteria.

Desenvolvimento e caracterização de polimerossomos para veiculação de L-asparaginase / Development and characterization of polymersomes for the release of L-asparaginase

A enzima L-Asparaginase (ASNase) é um biofámaco utilizado no tratamento da leucemia linfoblástica aguda, no entanto, a evolução da produção da ASNase como um medicamento desde o final da década de 1970 resultou em apenas quatro alternativas disponíveis no mercado farmacêutico, com relatos de graves reações imunogênicas e toxicidade. Desse modo, a nanotecnologia é uma plataforma que pode ser explorada para administração dessa enzima diminuindo a exposição da mesma a proteases e aumentando a sua meia-vida aparente. Os polimerossomos (PL) são opções que pela nanoestrutura vesicular poderiam encapsular a ASNase em seu core aquoso e pela presença de uma membrana polimérica, são mais robustos que os lipossomos. Assim, neste trabalho objetivou-se desenvolver PL para encapsulação da ASNase como uma alternativa às formulações deste biofármaco existentes. Foram desenvolvidos PL de PEG-PLA, PMPC-PDPA, PEG-PDPA e Pluronic® L-21. Foram estudados fatores relacionados à composição dos copolímeros (fração hidrofílica, responsividade a fatores externos tais como pH e temperatura) e métodos de elaboração (hidratação do filme polimérico, troca de pH e temperatura) bem como foi feita a caracterização dos PL obtidos (tamanho, índice de polidispersão, espessura de membrana, formação de excessivo bulk polimérico, obtenção de micelas). Também foi feito um planejamento racional para encapsulação da ASNase (hidratação direta do filme polimérico e encapsulação por eletroporação, autoagregação com encapsulação por troca de pH ou de temperatura). Para os PL preparados com PEG-PLA, a extrusão resultou em distribuição de tamanhos mais estreitos correspondentes aos valores de PDI de 0,345, 0,144 e 0,081 para PEG45-PLA69, PEG114-PLA153 e PEG114-PLA180, respectivamente. Foi demonstrado que copolímeros com menor fração hidrofóbica resultam em maior eficiência de encapsulação para proteínas, já que possuem volumes aquosos maiores. Com o PMPC25-PDPA72 foi possível encapsular em média três unidades de ASNase por vesículas através da eletroporação ou troca de pH, sendo que no primeiro método houve formação de túbulos e no último método as micelas não foram completamente removidas. Para PEG100-PDPA80, grandes agregados permaneceram após a purificação levando a um PDI alto, mas não foi observada a formação de túbulos, já a troca de pH para este copolímero resultou em maior perda de copolímeros como bulk polimérico precipitado. Para o copolimero tribloco Pluronic® L-121, foi observado que as vesículas eram estáveis durante uma semana à temperatura ambiente, contrariando o que era descrito na literatura. Nesses sistemas, quando preparados por hidratação do filme, a encapsulação da ASNase foi realizada por eletroporação mas a proteína não foi detectada dentro das vesículas. Atribuímos a não-encapsulação à organização da bicamada Pluronic® L-121 sem conformação definida das cadeias poliméricas, dificultando a reorganização do bloco hidrofílico na porção interna do poro durante eletroporação. Por troca de temperatura, cerca de 5 % de ASNase foi encapsulada e o método resultou em total recuperação da atividade da enzima. Desse modo foram obtidos diferentes PL com diferentes características nanoestruturais de acordo com os copolímeros utilizados para carreamento da ASNase

The enzyme L-Asparaginase (ASNase) is a biopharmaceutical used in the treatment of acute lymphoblastic leukemia, still the industrial production of ASNase as a marketable drug since the late 1970s has resulted in only four alternatives available in the pharmaceutical market, with reports of severe immunogenic reactions and toxicity. In this sense, nanotechnology is a platform that can be exploited to administer this enzyme by decreasing its exposure to proteases and increasing its apparent half-life. Polymerosomes (PL) are interesting routes which by its intrinsically vesicular nanostructure could encapsulate the ASNase in its aqueous core and by the presence of a polymeric membrane, being more robust than the liposomes. Thus, in this work it was intended to develop PL for ASNase encapsulation as an alternative to existing formulations of this biopharmaceutical. PL of PEG-PLA, PMPC-PDPA, PEG-PDPA and Pluronic® L-21 were developed. It was studied the copolymers composition (i.e. hydrophilic fraction, responsiveness to external factors such as pH and temperature), PL design (i.e. polymer film hydration, pH change and temperature) and PL characterization (i.e. size, polydispersity index - PDI, membrane thickness, formation of excessive polymer bulk, micelles production). A suitable experimental planning for ASNase encapsulation (i.e. direct hydration of the polymeric film and encapsulation by electroporation, self-aggregation with encapsulation by pH or temperature change) was also performed. For the PL prepared with PEG-PLA, the extrusion resulted in narrower size distribution corresponding to the PDI values of 0.345, 0.144 and 0.081 for PEG45-PLA69, PEG114-PLA153 and PEG114-PLA180, respectively. It has been shown that copolymers with lower hydrophobic fraction result in higher encapsulation efficiency for proteins, since they have larger aqueous volumes. With PMPC25-PDPA72 PL, it was possible to encapsulate three units of ASNase per vesicles through electroporation or pH change. In the first method, tubules were formed and in the latter one the micelles were not completely removed. For PEO100-PDPA80 PL, large aggregates remained after purification leading to a high PDI value, nevertheless no tubule formation was observed, since the pH change for this copolymer resulted in greater loss of copolymers as a precipitated polymer bulk. For the Pluronic® L-121 triblock copolymer PL, it was observed that the vesicles were stable for one week at room temperature, contrary to what was described in the literature. These PLs were prepared by film hydration method and ASNase encapsulation was performed by electroporation, nonetheless the protein was not detected within the vesicles. It is attributed the non-encapsulation to the organization of the Pluronic® L-121 bilayer without defined conformation of the polymer chains, making it difficult to reorganize the hydrophilic block in the internal portion of the pore during electroporation. By temperature change, about 5% of ASNase was encapsulated and the method resulted in complete recovery of enzyme activity. In conclusion, several PLs with a vast range of differential nanostructural characteristics were obtained according to the copolymers used for ASNase loading

Assessment of electrochemotherapy effects on the development of Ehrlich solid tumor in swiss mice using a novel electroporator device / Avaliação dos efeitos da eletroquimioterapia sobre o desenvolvimento do tumor sólido de Ehrlich em camundongos Swiss utilizando um novo eletroporador

Electrochemotherapy is a local anticancer treatment in which non-permeant chemotherapeutic drugs are associated with electric pulses of well-established parameters. The electric pulses cause pores to open on the plasma membrane and facilitate drug transport, enhancing cytotoxicity and reducing side effects. Assessment of electrochemotherapy effects on Ehrlich solid tumor development in this work aims to evaluate in vivo usage of the electroporator device developed by the Department of Electrical Engineering of Engineering School of UFMG. Therefore, 40 Swiss mice were inoculated with Ehrlich tumor cells, and developed the tumor in solid form. After 21 days, mice were subjected to specific treatment protocols (control, bleomycin, electric pulses and electrochemotherapy); 17 days later they were euthanized and the tumors collected for histopathology analysis. Electrochemotherapy induced discrete weight loss and an inflammatory response in the tumor, which was not seen on the other treatment groups. Bleomycin alone induced necrosis. Both groups showed lower cellular proliferation rates. From this study, it was concluded that the animals tolerated electrochemotherapy treatment under anesthesia and the electroporator device developed by the Engineering School of UFMG was adequate when used in an electrochemotherapy protocol.(AU)

Eletroquimioterapia é uma modalidade de tratamento local contra o câncer em que a administração de quimioterápicos não penetrantes à membrana plasmática é associada à aplicação de pulsos elétricos com parâmetros bem estabelecidos, que abrem poros na membrana plasmática e facilitam a entrada desses fármacos nas células, aumentando sua citotoxicidade e reduzindo efeitos colaterais. A avaliação dos efeitos da eletroquimioterapia sobre o desenvolvimento do tumor sólido de Ehrlich em camundongos Swiss neste trabalho teve como objetivo testar o uso in vivo do aparelho eletroporador desenvolvido pelo Departamento de Engenharia Elétrica da Escola de Engenharia da UFMG. Para tanto, foram utilizados 40 camundongos fêmeas da linhagem Swiss, nos quais foram inoculadas células de tumor de Ehrlich, para o desenvolvimento do tumor na forma sólida. Após 21 dias, os camundongos foram submetidos ao protocolo de tratamento específico (controle, bleomicina, pulsos elétricos e eletroquimioterapia); 17 dias depois foram eutanasiados e seus tumores coletados para análise histopatológica e imuno-histoquímica. A eletroquimioterapia induziu perda de peso discreta e uma resposta inflamatória no tumor que não foi observada nos outros grupos. O grupo bleomicina apresentou maior porcentagem de necrose. Ambos os grupos apresentaram menor índice de proliferação celular. Com este estudo, pode-se concluir que o tratamento sob anestesia foi bem tolerado pelos animais e que o aparelho eletroporador desenvolvido pela Escola de Engenharia da UFMG é adequado para utilização em um protocolo de eletroquimioterapia.(AU)

RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.

Utilização de eletroquimioterapia para carcinoma de células escamosas tegumentar em felino / Employment of electrochemotherapy for cutaneous squamous cell carcinoma in cats

A eletroquimioterapia compreende a utilização conjunta de fármacos antineoplásicos e aplicação regional de pulsos elétricos (eletroporação), maximizando a concentração intracelular destes fármacos, assim propiciando maior ação citotóxica. A bleomicina, fármaco antimicrobiano dotado de propriedade antineoplásica, apresenta restrita penetrabilidade na membrana celular, dada a sua hidrossolubilidade. Todavia, uma vez administrada via intralesional ou intravenosa associada à eletroporação, demonstra citotoxicidade potencializada. Foram utilizados 21 felinos acometidos por carcinoma de células escamosas tegumentar. Padronizou-se o protocolo eletroquimioterápico empregando-se sulfato de bleomicina, pela via intravenosa, na dose de 15U/m2 de superfície corpórea. A eletroporação foi perfilada com eletrodo composto por agulhas, pulsos elétricos com tensão de 1000 V, em onda quadrada unipolar, com duração de 100 microsegundos, totalizando oito ciclos. Verificou-se remissão neoplásica integral em 21 felinos inclusos no estudo (100%). Inexistiram complicações e/ou efeitos adversos decorrentes do procedimento. O protocolo avaliado neste trabalho revelou-se exequível, eficaz e seguro na terapêutica antineoplásica de carcinoma de células escamosas tegumentar felino.

Electrochemotherapy is characterized as a protocol which combines the use of antineoplastic agents and localized application of electric pulses (electroporation) to improve the intracellular concentration of these agents, increasing its cytotoxic action. Bleomycin, an antibiotic agent with antineoplastic properties, is a hydrophilic molecule, having a restricted transport through the cellular membrane. However, when it is administered intralesionally or intravenously and associated to electroporation, its cytotoxicity is maximized. There were utilized 21 cats affected by cutaneous squamous cell carcinoma. The electrochemotherapy protocol was standardized using intravenous bleomycin sulfate at a dose of 15U/m2 body surface area. Electroporation was performed using an electrode composed of needles and electric pulses with 1000 V voltage, in unipolar square wave and 100 microseconds duration, totalizing eight cycles. There was complete neoplastic remission in 21 cats (100%). There were no complications or side effects associated with the procedure. The protocol studied in this work showed to be feasible, effective and safe for antineoplastic therapy in feline cutaneous squamous cell carcinoma.

Processo autofágico no reconhecimento de Escherichia coli enteroinvasora: um possível mecanismo da degradação bacteriana por células epiteliais / Autophagy in recognition of Enteroinvasive Escherichia coli: a possible bacterial degradation mechanism by epithelial cells

O reconhecimento de bactérias invasoras pelas células hospedeiras através do processo autofágico é um fator chave na determinação da infecção bacteriana. Escherichia coli enteroinvasora (EIEC) possui uma proteína, denominada IcsB, que em estudos em Shigella, é responsável pela inativação deste processo de degradação bacteriana. Uma vez que EIEC expressa menos IcsB do que S. flexneri, nos propusemos a investigar o processo autofágico na infecção por EIEC, utilizando as técnicas de mutação gênica por inserção, western-blot, microscopia de fluorescência e eletrônica de transmissão e microarray. Verificamos que a proteína IcsB é um fator de virulência importante na camuflagem de EIEC, pois quando pouco ou nada expresso, há um maior reconhecimento da bactéria pelas células hospedeiras, favorecendo sua menor disseminação. Isto corrobora não somente com a transcrição gênica, mas com a importância da sequência de nucleotídeos deste gene, uma vez que a cepa de E. coli SM124/13 complementada com o icsB de Shigella se mostrou mais eficiente na disseminação dentro da célula hospedeira. De forma interessante, IcsB apresentou um papel inédito na regulação da resposta inflamatória das células HeLa, onde a ausência de IcsB em EIEC promoveu uma intensa perturbação na homeostase da célula hospedeira, com aumento da secreção de IL-6, IL-8 e morte celular. Adicionalmente, ficou evidente que a célula eucariótica responde de maneira distinta frente a infecção por EIEC e Shigella flexneri. EIEC provavelmente ativou o processo autofágico em células humanas de forma não canônica. Nossa hipótese seria de que EIEC é reconhecida pelo processo autofágico, podendo ser este um importante fenômeno de reconhecimento bacteriano que colabore para a menor disseminação intracelular de EIEC, e assim tornar sua doença mais branda, quando comparada com a infecção por Shigella

The invasive bacteria recognition by host cells through autophagy is a key factor for determining bacterial infection. Enteroinvasive Escherichia coli (EIEC) express a protein IcsB, which in Shigella, is known for inactivating the bacterial degradation process. Once EIEC showed less expression of icsB when compared to S. flexneri, we proposed to investigate the autophagy caused by EIEC infection, using techniques such as gene mutation by insertion, western blot, fluorescence microscopy, transmission electron microscopy and microarray. Our results showed that IcsB protein is an important virulence factor in EIEC because it causes a camouflage of the bacteria in the eukaryotic cell. When there is a low expression of the protein, the cell recognition of the invasive bacteria is high, decreasing the bacteria dissemination. This found confirms the importance of the gene transcription and the gene sequence, since the strain E. coli SM124/13, complemented with icsB from Shigella, showed higher dissemination efficiency inside of the host cell. Interestingly, IcsB showed a new role on regulating the inflammatory response in Hela cells. The absence of IcsB in EIEC generated an intense disturbance of the cell homeostasis, increased the secretion of IL-6 and IL-8, and caused cell death. Additionally, our results revealed that eukaryotic cell infected by EIEC or Shigella flexneri showed distinguish responses. In EIEC infection, the autophagy was activated in human cells, but not in a conventional mode. Our hypothesis is that EIEC is recognized by autophagy, being an important cell process for bacterial recognition. This process can cause a decrease in the intracellular spread of EIEC making the infection less severe when compared to the infection caused by Shigella

Use of green fluorescent protein to monitor Lactobacillus plantarum in the gastrointestinal tract of goats

The experiment aimed to specifically monitor the passage of lactobacilli in vivo after oral administration. The green fluorescent protein (GFP) gene was cloned downstream from the constitutive p32 promoter from L. lactis subsp. cremoris Wg2. The recombinant expression vector, pLEM415-gfp-p32, was electroporated into Lactobacillus plantarum (L. plantarum) isolated from goat. Green fluorescent protein (GFP) was successfully expressed in L. plantarum. After 2 h post-administration, transformed Lactobacillus could be detectable in all luminal contents. In the rumen, bacteria concentration initially decreased, reached the minimum at 42 h post-oral administration and then increased. However, this concentration decreased constantly in the duodenum. This result indicated that L. plantarum could colonize in the rumen but not in the duodenum.

A detailed description of an economical setup for electroporation of chick embryos in ovo

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.

Fosfoproteômica e proteômica quantitativa de células mesenquimais durante a diferenciação osteoblástica mediada por BMP2, expressão e purificação de diferentes tipos de proteínas morfogenéticas ósseas / Quantitative phosphoproteomics and proteomics of mesenchymal stem cells during BMP2-mediated osteoblastic differentiation, expression and purification of different types of bone morphogenetic proteins

As fraturas e perdas ósseas representam altos riscos para o Sistema público de Saúde (SUS), além de afetar a qualidade de vida do paciente, portanto é necessário o entendimento das bases moleculares que envolvem os mecanismos de reparo ósseo. Citocinas secretadas por células do sistema imune presentes no local da inflamação, como as IL-6, IL-10 e TNFα atuam como fatores quimiotáticos para células mesenquimais, que proliferam e se diferenciam em osteoblastos pela ação autócrina e parácrina de Proteínas Morfogenéticas Ósseas (BMPs), principalmente a BMP2. Embora seja conhecido que a ação de BMP2 ocorra através de sua ligação nos receptores ActRI/BMPR, que ativam proteínas SMADS 1/5/8 efetoras, pouco se sabe sobre os mecanismos intracelulares que participam do processo de diferenciação osteoblástico. Neste estudo propôs-se analisar as diferenças no conteúdo de proteínas totais e de proteínas fosforiladas em células mesenquimais de pele induzidas à osteogênese pelo tratamento com BMP2 por diferentes períodos de tempo, utilizando-se de Isótopos Estáveis de Dimetila acoplado ao LC/MS. A partir de 150µg de material inicial, foi possível identificar 2.264 proteínas, as quais foram quantificadas nos diferentes pontos de indução, sendo que 235 são fosforiladas. Análise de motivos de quinases mostrou que diversos substratos possuem sítios fosforilados correspondentes àqueles dos motivos de fosforilação das quinases Casein Kinase, p38, CDK e JNK. A análise da ontologia gênica mostrou um aumento de processos biológicos relacionados com sinalização e diferenciação após a primeira hora de indução com rhBMP2. Além disso, proteínas envolvidas com o rearranjo do citoesqueleto e com vias de sinalização Wnt e Ras foram encontradas como tendo fosforilação diferencial durante todos os períodos estudados. Os dados revelaram novos substratos intracelulares que são fosforilados nos primeiros momentos do comprometimento com a diferenciação osteoblástica mediada pelo tratamento com rhBMP2 em células mesenquimais derivadas da pele. Além disso, clones celulares que superexpressam as proteínas recombinantes humanas BMP2 e BMP4 foram gerados, e sua atividade verificada in vitro. Paralelamente, a rhBMP7, obtida anteriormente, foi purificada por cromatografia de afinidade utilizando-se uma coluna de Heparina-Sepharose, que foi posteriormente utilizada para ensaios in vitro e in vivo, nos quais se mostrou capaz de gerar osteoblastos e tecido ósseo, respectivamente, o que abre novas possibilidades para o uso destas proteínas como biofármacos no Brasil

Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated human skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. From 150 µg of starting material, 2,264 proteins containing two or more peptides were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during commitment to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. Cell clones overexpressing the human BMP 2 and 4 recombinant proteins were also generated, and their biological activity was confirmed in vitro. In parallel, chromatography-affinity purified rhBMP7, obtained using heparin-Sepharose columns, was used for in vivo and in vitro assays to evaluate the ability of this purified protein to generate osteoblasts and bone tissue, respectively, opening new avenues for the use of these proteins as biopharmaceuticals in Brazil

Enhancing DNA electrotransformation efficiency in escherichia coli DH10B electrocompetent cells

Electrotransformation also known as electroporation is the most reliable and efficient tool for plasmid DNA uptake. Electrotransformation efficiency is function of many factors which include (1) number of cell washes prior to electroporation, (2) electroporation cell number, (3) electroporation DNA amount, and (4) cell growth phase. Those factors have limitedly been concomitantly investigated in E. coli DH10B strain. This study is aimed to explore above key factors to define the optimal conditions for high electrotransformation efficiency. The results showed that electrotransformation efficiency of E. coli DH10B was enhanced to 1.5 x 10(9) cfu/ug by washing cells three times with 15 ml of 10 percent glycerol. This washed off extra salts from cell suspension and enhanced electrotransformation by preventing arcing and enhancing cell resistance while ensuring minimal level of conductivity. Early exponential phase at 0.15 OD600 was the best growth phase for enhancing electrotransformation of E. coli DH10B. The results also showed that higher electrotransformation efficiency was similarly achieved when 0.5 x 10(10) and 0.6 x 10(10) cell numbers were electroporated with DNA amount ranging from 10 to 40 pg. This study confirmed the optimal conditions for electro competent cell preparation and plasmid DNA electrotransformation, which can result highest transformation efficiency.

Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens

Electroporation entails brief, high intensity pulse to create transient pores in the cell membrane to facilitate the entry of exogenous macromolecules, which may otherwise be excluded. Removal of the external field leads to the resealing of the membrane electropores permitting the survival of the electrically stimulated recipient cells. Using this technique foreign deoxyribonucleic acid (DNA) has been successfully introduced into many cell types both from prokaryotes and eukaryotes. Increase in pulse voltage and length beyond a critical limit has been reported to decrease transformation efficiency, hence in this study we have investigated another strategy i.e. increase in the number of pulses at constant high voltage and pulse duration. Commonly used Agrobacterium strains LBA4404 and EHA101 and binary vector pCAMBIA1301 were used. Transformants were selected on a combination of hygromycin and kanamycin, and confirmed by polymerase chain reaction (PCR) and restriction analysis. Increase in the number of pulses was found to show a significant and linear increase in transformation efficiency.

An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains

This paper describes an efficient bacterial transformation system that was established for the preparation of competent cells, plasmid preparation, and for the storage in bacterial stocks in our laboratory. Using this method, a number of different plasmids have been amplified for further experiments. Competent cells for bacterial transformation were prepared by the calcium chloride method with an optimum concentration of 75 mM. Three different strains of Escherichia coli that were tested are DH5 alpha, TG1 and XL1 blue, and the most efficient strain being XL1 blue. The optimal optical density (OD600) range for competent cell preparation varied for each of the strains investigated, and for XL1 blue it was 0.15-0.45; for TG1 it was 0.2-0.5; and for DH5 alpha it was 0.145-0.45. The storage time of competent cells and its correlation to transformation efficiency has been studied, and the result showed that competent cells can be stored at –20 ºC for 7 days and at –70 ºC for 15 days. Three critical alterations to previous methods have been made, which are the changing of the normal CaCl2 solution to TB solution, the changing of the medium from LB to S.O.C., and addition of DMSO or PEG8000 during transformation of competent cells with plasmids. Changing the medium from LB to S.O.C., resulted in much faster growth of transformants, and the transformation efficiency was increased. Addition of DMSO or PEG8000 raised transformation efficiencies by 100-300 fold. Our improved bacterial transformation system can raise the transformation efficiency about 103 times, making it becoming a highly efficient bacterial transformation system.

Administração do gene da proteína verde fluorescente no músculo tireoaritenóideo do rato pela técnica de eletroporação / Administration of the green fluorescent protein gene into the rat thyroarytenoid muscle using the electroporation technique

A eletroporação é uma técnica de administração de pulsos elétricos no tecido que promove a entrada de genes na célula. Utilizou-se o gene do marcador da proteína fluorescente verde, o qual foi injetado no músculo tireoaritenóideo previamente à aplicação dos pulsos. Foram estudadas 32 laringes de ratos, divididas em grupos de alta voltagem, baixa voltagem e associação de alta e baixa voltagem. Cortes histológicos foram analisados e os grupos comparados quanto a número, intensidade da fluorescência, distribuição das fibras musculares marcadas e infiltrado inflamatório. O grupo de alta e baixa voltagens foi o mais eficiente, de acordo com os parâmetros estudados.Electroporation is a technique for the application of electric pulses in a tissue that allows the entrance of genes into a cell. The gene Enhanced green fluorescent protein was injected into the thyroarythenoid muscle before the pulse application. Thirty- two rat larynges were divided into groups of high voltage, low voltage and high and low voltage together. Histological sections were analized and the groups were compared in relation to the number, fluorescent signal, distribution of the marked muscular fibers and the inflammatory pattern. The group of high and low voltage pulses had the best performance according to these parameters...

Establecimiento de un método para la transformación genética de café (coffea arabica cv. catimor) e incorporación del gen bar que confiere resistencia al herbicida glufosinato de amonio / Establisment of a genetic transformation method of coffee (coffea arabica cv. catimor) and incorporation of bar gene for ammonium

Con el fin de establecer un método de transformación genética para Coffea arabica cv. Catimor, se evaluaron las condiciones óptimas de regeneración y transformación por electroporación en diferentes tejidos de la planta. Se optimizó el sistema de células en suspención, utilizando una sola hormona (BA), obteniéndose altos rendimientos de embriones somáticos primarios y secundarios. Igualmente se estableció el método del MTT (1 por ciento) y las dosis de glufosinato de amomio para la selección de células viables y células potencialmente transformadas (1 mg/L para hoja, callo y embriones somáticos y 5 mg/L para células) respectivamente. Se electroporaron distintos tejidos (calo embriogénico, hojas de vitroplantas, embriones globulares y torpedo), con distintas condiciones, encontrándose el mayor porcentaje de expresión transitoria del gen gus por explante, en tejidos pre-tratados enzimáticamente, a 375 V/cm en callos, y 625 V/cm en hojas y embriones. Los únicos tejidos electroporados que regeneraron plantas, fueron las secciones de embriones somáticos tipo torpedo cultivados en medio líquido, que dieron orígen a embriones somáticos secundarios. Dichos embriones somáticos resultaron positivos en el ensayo histoquímico para el gen gus y en la reacción de PCR para los genes gus y bar

Promotores de permeaçäo para a liberaçäo transdérmica de fármacos: uma nova aplicaçäo para as ciclodextrinas / Permeation enhancers in transdermal drug delivery systems: a new aplication of cyclodextrins

No presente trabalho é feita uma breve revisäo sobre promotores de permeaçäo cutânea, descrevendo-se os seus mecanismos de açäo e alguns exemplos. Abordam-se as vias de permeaçäo de fármacos através da pele e liberaçäo transdérmica. Säo também focadas as ciclodextrinas e seu derivados, a sua estrutura e propriedades físico-químicas, formaçäo de complexos de inclusäo e o seu papel como excipientes em sistemas transdérmicos. As ciclodextrinas constituem um grupo de excipientes que têm um papel de grande importância em formulaçäo farmacêutica. Uma das mais extradionárias propriedades destas moléculas é a sua capacidade de incrementar a liberaçäo de fármacos através da pele sem, no tentanto, afetar a sua funçäo barreira...

Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl.) Sw. protoplasts

In order to develop a high-efficiency and reproducible transformation protocol for Stylosanthes guianensis we assessed the biological and physical parameters affecting plant electroporation protoplasts. Energy input, as combinations of electric field strengths discharged by different capacitors, electroporation buffer and DNA form were evaluated. Transformation efficiency was assayed in vivo as transient reporter gene expression, using the GFP-coding gene mgfp5 driven by a CaMV 35S constitutive promoter. Energy input and electric field strength had a critical influence on transgene expression with higher transformation levels being achieved with 250 V.cm-1 discharged by 900 and 1000 muF capacitors. Linear plasmid DNA, the absence of chloride and the presence of calcium ions also increased transient gene expression, albeit not significantly