SodERF3, a novel sugarcane ethylene responsive factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants.

The molecular signals and pathways that govern biotic and abiotic stress responses in sugarcane are poorly understood. Here we describe SodERF3, a sugarcane (Saccharum officinarum L. cv Ja60-5) cDNA that encodes a 201-amino acid DNA-binding protein that acts as a transcriptional regulator of the ethylene responsive factor (ERF) superfamily. Like other ERF transcription factors, the SodERF3 protein binds to the GCC box, and its deduced amino acid sequence contains an N-terminal putative nuclear localization signal (NLS). In addition, a C-terminal short hydrophobic region that is highly homologous to an ERF-associated amphiphilic repression-like motif, typical for class II ERFs, was found. Northern and Western blot analysis showed that SodERF3 is induced by ethylene. In addition, SodERF3 is induced by ABA, salt stress and wounding. Greenhouse-grown transgenic tobacco plants (Nicotiana tabacum L. cv. SR1) expressing SodERF3 were found to display increased tolerance to drought and osmotic stress.

Biorreactores de un solo uso en la industria biofarmacéutica y su uso en la producción de candidatos vacunales contra SARS- COV-2. Artículo de revisión / Single-use systems bioreactors in the biopharmaceutical industry and its use in SARS-CoV-2 vaccine's candidates production. A review

Los biorreactores de sistemas de un solo uso (SUSs), también conocidos como biorreactores desechables, se han convertido en una parte integral de las instalaciones biotecnológicas de fabricación para bioproductos con un mercado potencial que espera una tasa de crecimiento de casi el 15,5% durante el período pronosticado: 2018 a 2023. Los biorreactores SUSs son más seguros, simples y flexibles al compararlos con sus contrapartes, biorreactores de acero inoxidable, por lo que su uso se está incrementando en la industria biofarmacéutica principalmente en la planificación de vías rápidas de proyectos complejos, incluidos los relacionados con la pandemia de SARS-CoV-2. Así, el uso de SUS se ha convertido en una alternativa eficaz para la producción rápida de candidatos a vacunas. Pero algunas desventajas técnicas y operativas aún obstaculizan su uso en todo el mundo. Esta revisión brinda una visión racional del uso, los tipos, los parámetros operativos y las nuevas aplicaciones de los biorreactores SUSs en la industria biofarmacéutica. Asimismo, también se discuten los parámetros apropiados y las limitaciones de este equipo, enfocándose en su uso para la producción de vacunas contra COVID-19

Single-Use-Systems (SUSs) Bioreactors, also known as disposable bioreactors, have become an integral part of biotechnology manufacturing facilities for bioproducts with a potential market expecting a growth rate of nearly 15.5% over the forecast period: 2018 to 2023. SUSs bioreactors are comparatively safe, simple, and flexible than their stainless-steel bioreactors counterparts thus, their usage is being augmented in the biopharmaceutical industry mainly in planning fast tracks of complex projects, including those related to the SARS-CoV-2 pandemic. Thus, the use of SUSs has become an effective alternative for the rapid production of vaccine candidates. However, some technical and operational disadvantages still hamper their worldwide use. This review gives a rational insight into SUSs bioreactors use, types, operational parameters and new applications in the biopharmaceutical industry. Likewise, the appropriate parameters and limitations of this equipment, focusing on its use for vaccine production against COVID-19 are also discussed

A protocol for the purification of bovine serum albumin free of deoxyribonuclease activity.

Bovine serum albumin, free of deoxyribonuclease activity, was obtained in our laboratory using ion-exchange chromatography followed by acetylation. Chromatography on four different resins (DEAE-52, P-11, hydroxylapatite and Q Sepharose fast-flow) was examined. Fractions from Q Sepharose chromatography, eluted with a linear gradient 0-1.0 M NaCl and subsequently acetylated, proved to be the most effective method for obtaining deoxyribonuclease-free bovine serum albumin.

Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes.

We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assays results in a real cloning experience, we performed a cloning simulation assay using the same conditions established for the radioactive assay (enzyme units and pmols of DNA ends). As a result, we found that for degradation percentages of the radioactive DNA substrate per enzyme unit below 0.5, the false positives in the cloning stimulation assay were less than 5%. This conditions could ensure a good performance of the enzyme preparations for cloning experiments. Finally, we described the use of the radiolabeled [gamma 33P] ATP lambda Hpa II DNA substrate to detect 5'-->3' single stranded DNA dependent exonuclease and phosphatase contaminating activities in some critical steps of the purification process of the restriction enzyme Kpn I.

Fructo-oligosaccharides production by the Gluconacetobacter diazotrophicus levansucrase expressed in the methylotrophic yeast Pichia pastoris.

Levansucrase (LsdA) (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (formerly Acetobacter diazotrophicus) yields high levels of fructo-oligosaccharides (FOS) from sucrose. A DNA fragment encoding the precursor LsdA lacking the first 57 amino acids was fused to the pho1 signal sequence under the control of the Pichia pastoris-alcohol oxidase 1 (AOX1) promoter. Methanol induction of a P. pastoris strain harboring a single copy of the lsdA expression cassette integrated in the genome resulted in the production of active levansucrase. After fermentation of the recombinant yeast, LsdA activity was detected in the periplasmic fraction (81%) and in the culture supernatant (18%) with an overall yield of 1% of total protein. The recombinant LsdA was glycosylated and displayed optimal pH and temperature for enzyme activity similar to those of the native enzyme, but thermal stability was increased. Neither fructosylpolymerase activity nor FOS production was affected. Incubation of recombinant LsdA in sucrose (500 g l(-1)) yielded 43% (w/w) of total sugar as 1-kestose, with a conversion efficiency about 70%. Intact recombinant yeast cells also converted sucrose to FOS although for a 30% efficiency.

Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids.

Zinc and imidazole salts were applied for the detection of nucleic acids on either polyacrylamide of agarose gels. After electrophoresis, polyacrylamide gels are washed in distilled water to remove most of the residual electrophoresis reagents, then incubated in 10 mM zinc sulfate for 10 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colorless bands. Staining of nucleic acids on agarose gels can be performed by incubation in 40 mM zinc sulfate for 10 min, followed by immersion in 0.2 M imidazole for 5 min to form a deep white-stained background. On soaking in 2 M imidazole for 45 min, the imidazole-induced zinc precipitate is removed from the positions were nucleic acids are located resulting in a negative image of colorless and transparent nucleic acid bands against a white background. The sensitivity of this stain ranges from 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/band for different 22-base oligonucleotides, from 62 to 125 ng/band for large (from 20 to 2 kbp) DNA, and is 1 microgram/band for human peripheral-blood monocyte RNA. After chelation of zinc with EDTA, the nucleic acids can be quantitatively recovered from the gel. The principal advantage of this technique over ethidium bromide staining is evident for preparative purposes. Using zinc-imidazole in the detection of purified pBACIB.1 (2.8 kbp) plasmid DNA and anti-HBsAg single chain Fv antibody fragment (0.7 kbp) DNA, followed by elution from gel slices, ligation and transformation of competent E. coli XL-1 Blue cells, the number of transformants notably increased from 280 (obtained with conventional ethidium bromide staining plus UV-irradiation at 312 nm) to 10,000.

Purificación de la enzima de restricción Sma I / Purification of Sma I restriction enzyme

La enzima de retricción Sma I, producida por el microorganismo Serratia marcescens, ha sido purificada en nuestro laboratorio utilizando precipitación con PEG 6000 y cromatografía de intercambio iónico en fosfocelulosa (P-11 Whatman), obteniéndose una preparación final con una actividad específica de 2971 U/mg proteína y 35 % de recobrado total del proceso de purificación apta para ser utilizada en los experimentos de clonación y otras técnicas del ADN recombinante

Purificación de la enzima de restricción NciI / Purification of NciI restriction endonuclease

La enzima de restricción Nci I producida por el microorganismo Neisseria cinerea, se purificó en nuestro laboratorio utilizando cromatografía de afinidad y de intercambio iónico, lo que permitió obtener una preparación enzimática con una actividad específica de 20 000 U/mg de proteínas y 69,2 % de recobrado global del proceso apta para ser usada en los diferentes trabajos relacionados con la ingeniería genética