Milligram scale production of potent recombinant small interfering RNAs in Escherichia coli.

Abstract:

:Small interfering RNAs (siRNAs) are invaluable research tools for studying gene functions in mammalian cells. siRNAs are mainly produced by chemical synthesis or by enzymatic digestion of double-stranded RNA (dsRNA) produced in vitro. Recently, bacterial cells, engineered with ectopic plant viral siRNA binding protein p19, have enabled the production of "recombinant" siRNAs (pro-siRNAs). Here, we describe an optimized methodology for the production of milligram amount of highly potent recombinant pro-siRNAs from Escherichia coli cells. We first optimized bacterial culture medium and tested new designs of pro-siRNA production plasmid. Through the exploration of multiple pro-siRNA related factors, including the expression of p19 protein, (dsRNA) generation method, and the level of RNase III, we developed an optimal pro-siRNA production plasmid. Together with a high-cell density fed-batch fermentation method in a bioreactor, we have achieved a yield of ~10 mg purified pro-siRNA per liter of bacterial culture. The pro-siRNAs produced by the optimized method can achieve high efficiency of gene silencing when used at low nanomolar concentrations. This new method enables fast, economical, and renewable production of pure and highly potent bioengineered pro-siRNAs at the milligram level. Our study also provides important insights into the strategies for optimizing the production of RNA products in bacteria, which is an under-explored field.

journal_name

Biotechnol Bioeng

authors

Kaur G,Cheung HC,Xu W,Wong JV,Chan FF,Li Y,McReynolds L,Huang L

doi

10.1002/bit.26740

subject

Has Abstract

pub_date

2018-09-01 00:00:00

pages

2280-2291

issue

9

eissn

0006-3592

issn

1097-0290

journal_volume

115

pub_type

杂志文章
  • Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass.

    abstract::A semimechanistic multi-reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose-to-glucose and two heterogeneous reactions of cellulose-to-cellobiose and cell...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.22197

    authors: Zheng Y,Pan Z,Zhang R,Jenkins BM

    更新日期:2009-04-15 00:00:00

  • Effect of pH change on the performance and microbial community of enhanced biological phosphate removal process.

    abstract::An acetate-rich wastewater, containing 170 mg/L of total organic carbon (TOC), 13 mg/L of N, and 15 mg/L of P, was treated using the enhanced biological phosphate removal (EBPR) process operated in a sequencing batch reactor. A slight change of pH of the mixed liquor from 7.0 to 6.5 led to a complete loss of phosphate...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.20589

    authors: Zhang T,Liu Y,Fang HH

    更新日期:2005-10-20 00:00:00

  • Online monitoring of microcarrier based fibroblast cultivations with in situ microscopy.

    abstract::Animal cell culture is widely used in biotechnology for the production of many biological products. In situ microscopes acquire images directly from cell suspensions and analyze the images in matters of cell concentration, cell size distribution and cell morphology. Their applicability was already proven for yeast and...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.21523

    authors: Rudolph G,Lindner P,Gierse A,Bluma A,Martinez G,Hitzmann B,Scheper T

    更新日期:2008-01-01 00:00:00

  • Enhanced acetone-butanol fermentation using repeated fed-batch operation coupled with cell recycle by membrane and simultaneous removal of inhibitory products by adsorption.

    abstract::A novel acetone-butanol production process was developed which integrates a repeated fed-batch fermentation with continuous product removal and cell recycle. The inhibitory product concentrations of the fermentation by Clostridium acetobutylicum were reduced by the simultaneous extraction process using polyvinylpyridi...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.260470405

    authors: Yang X,Tsao GT

    更新日期:1995-08-20 00:00:00

  • Increased heterologous protein production by Saccharomyces cerevisiae growing on ethanol as sole carbon source.

    abstract::Saccharomyces cerevisiae is a widely used host organism for the production of heterologous proteins, often cultivated in glucose-based fed-batch processes. This production system however has many factors limiting the productivity, mainly towards the end of the fermentation. For the optimised production of a Camelid an...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.21150

    authors: van de Laar T,Visser C,Holster M,López CG,Kreuning D,Sierkstra L,Lindner N,Verrips T

    更新日期:2007-02-15 00:00:00

  • Mechanistic insights into the liquefaction stage of enzyme-mediated biomass deconstruction.

    abstract::Effective enzyme-mediated viscosity reduction, disaggregation, or "liquefaction," is required to overcome the rheological challenges resulting from the fibrous, hygroscopic nature of lignocellulosic biomass, particularly at the high solids loadings that will be required for an economically viable process. However, the...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.26381

    authors: van der Zwan T,Hu J,Saddler JN

    更新日期:2017-11-01 00:00:00

  • Redox control bioreactor: a unique biological water processor.

    abstract::The redox control bioreactor (RCB) is a new hollow fiber membrane bioreactor (HFMBR) design in which oxygen and hydrogen gases are provided simultaneously through separate arrays of juxtaposed hollow fiber (HF) membranes. This study applied the RCB for completely autotrophic conversion of ammonia to N(2) through nitri...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.21617

    authors: Smith DP,Rector T,Reid-Black K,Hummerick M,Strayer R,Birmele M,Roberts MS,Garland JL

    更新日期:2008-03-01 00:00:00

  • Effect of heat on phycoerythrin fluorescence: Influence of thermal exposure on the fluorescence emission of R-phycoerythrin.

    abstract::The goal of this work was to measure and model the effect of thermal exposure on the fluorescence emission of R-phycoerythrin (R-PE). The long-term objective of our work is to assess the feasibility of encapsulating R-PE for use as the critical component of a time-temperature integrator (TTI) for ascertaining the degr...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.10686

    authors: Vaidya S,Orta-Ramirez A,Smith DM,Ofoli RY

    更新日期:2003-08-20 00:00:00

  • External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation.

    abstract::Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:

    authors: Quong D,Neufeld RJ,Skjåk-Braek G,Poncelet D

    更新日期:1998-02-20 00:00:00

  • Simulations of host-plasmid interactions in Escherichia coli: Copy number, promoter strength, and ribosome binding site strength effects on metabolic activity and plasmid gene expression.

    abstract::A mechanistically detailed single-cell model E. coli B/r-A was adapted to simulate the effects of vector presence on cell metabolism. Competition for RNA polymerase between chromosome and plasmid DNA is explicitly included. Distribution of active ribosomes among chromosome- and plasmid-derived messenger RNA, another k...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.260290305

    authors: Peretti SW,Bailey JE

    更新日期:1987-02-01 00:00:00

  • Improvement of product yields by temperature-shifting of Escherichia coli cultures containing plasmid pOU140.

    abstract::Temperature shifting was investigated as a means of improving cloned-gene product yields form a recombinant Escherichia coli containing the temperature-sensitive plasmid, pOU 140. In a series of shaker flask fermentations recombinant cells were thermally induced for different time periods. The growth, stability, and p...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.260290416

    authors: Dipasquantonio VM,Betenbaugh MJ,Dhurjati P

    更新日期:1987-03-01 00:00:00

  • High-throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell culture.

    abstract::Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell cultur...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.26839

    authors: Nierode GJ,Gopal S,Kwon P,Clark DS,Schaffer DV,Dordick JS

    更新日期:2019-01-01 00:00:00

  • A CRISPR-Cas12a-Derived Biosensor Enabling Portable Personal Glucose Meter Readout for Quantitative Detection of SARS-CoV-2.

    abstract::Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly throughout the whole world and caused significant difficulties in the prevention and control of the epidemic. In this case, several detection methods have been established based on nucleic acid diagnostic techniques and immunoassays to ach...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.27673

    authors: Huang D,Shi Z,Qian J,Bi K,Fang M,Xu Z

    更新日期:2021-01-06 00:00:00

  • Hollow-fiber membrane bioreactors using immobilized E. coli for protein synthesis.

    abstract::Actively growing Escherichia coli C600(pBR322), immobilized within the macroporous matrix of asymmetric-wall hollow-fiber membranes, has been propagated to extremely high densities, typically more than 10(12) cells/mL of accessible void volume, in some regions cells accounting for nearly 100% of the available macrovoi...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.260251113

    authors: Inloes DS,Smith WJ,Taylor DP,Cohen SN,Michaels AS,Robertson CR

    更新日期:1983-11-01 00:00:00

  • Oxygen-responsive genetic circuits constructed in Synechocystis sp. PCC 6803.

    abstract::As photoautotrophic prokaryotes, cyanobacteria are promising platforms for producing value-added bioproducts. However, few regulatory genetic parts and devices (e.g., inducible promoters and regulatory circuits) have been developed for these potential hosts. Furthermore, the devices that have been created respond only...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.25722

    authors: Immethun CM,Ng KM,DeLorenzo DM,Waldron-Feinstein B,Lee YC,Moon TS

    更新日期:2016-02-01 00:00:00

  • Kinetic modeling of oligosaccharide synthesis catalyzed by leuconostoc mesenteroides NRRL B-1299 dextransucrase

    abstract::The kinetic behavior of soluble and insoluble forms of dextransucrase from Leuconostoc mesenteroides NRRL B-1299 was investigated with sucrose as substrate and maltose as acceptor. To study the parameters involved, a kinetic model was applied that was previously developed for L. mesenteroides NRRL B-512F dextransucras...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/(sici)1097-0290(19990505)63:3<308::aid-bit

    authors: Dols M,Remaud-Simeon M,Willemot RM,Demuth B,Jordening HJ,Buchholz K,Monsan P

    更新日期:1999-05-05 00:00:00

  • Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions.

    abstract::The effects of ZnCl2 additions on a mercuric reductase, merA, ammonia monooxygenase, amoA, and hydroxylamine (NH2OH) oxidoreductase, hao, gene expression were examined in continuously cultured Nitrosomonas europaea cells. The reactor was operated for 85 days with a 6.9 d hydraulic retention time and with four successi...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.22069

    authors: Radniecki TS,Semprini L,Dolan ME

    更新日期:2009-02-01 00:00:00

  • Cell-surface exposure of a hybrid 3-cohesin scaffoldin allowing the functionalization of Escherichia coli envelope.

    abstract::Cellulosomes are large plant cell wall degrading complexes secreted by some anaerobic bacteria. They are typically composed of a major scaffolding protein containing multiple receptors called cohesins, which tightly anchor a small complementary module termed dockerin harbored by the cellulosomal enzymes. In the presen...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.27242

    authors: Vita N,Borne R,Fierobe HP

    更新日期:2020-03-01 00:00:00

  • Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance.

    abstract::In order to evaluate the sensitivity of hepatocellular cultures to variations in both substrate stiffness and bioactive ligand presentation, hepatocytes were cultured on differentially compliant polyacrylamide gel discs functionalized with varying amounts of the ECM ligand, fibronectin (FN). Subconfluent cell cultures...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.20328

    authors: Semler EJ,Lancin PA,Dasgupta A,Moghe PV

    更新日期:2005-02-05 00:00:00

  • Microbial granulation for lactic acid production.

    abstract::This work investigated the formation of microbial granules to boost the productivity of lactic acid (LA). The flocculated form of LA-producing microbial consortium, dominated by Lactobacillus sp. (91.5% of total sequence), was initially obtained in a continuous stirred-tank reactor (CSTR), which was fed with 2% glucos...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.25540

    authors: Kim DH,Lee MK,Hwang Y,Im WT,Yun YM,Park C,Kim MS

    更新日期:2016-01-01 00:00:00

  • The denitrification capability of cluster 1 Defluviicoccus vanus-related glycogen-accumulating organisms.

    abstract::The denitrification capability of Cluster 1 Defluviicoccus vanus-related glycogen-accumulating organisms (DvGAOs) is investigated. A sequencing batch reactor (SBR) fed with acetate as the sole carbon source was operated under alternating anaerobic-aerobic conditions to enrich Cluster 1 DvGAOs. Fluorescence in situ hyb...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.21711

    authors: Wang X,Zeng RJ,Dai Y,Peng Y,Yuan Z

    更新日期:2008-04-15 00:00:00

  • Effects of culture conditions on N-glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells.

    abstract::CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein thera...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.22644

    authors: Borys MC,Dalal NG,Abu-Absi NR,Khattak SF,Jing Y,Xing Z,Li ZJ

    更新日期:2010-04-15 00:00:00

  • A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.

    abstract::A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycopr...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.26291

    authors: Yin B,Wang Q,Chung CY,Bhattacharya R,Ren X,Tang J,Yarema KJ,Betenbaugh MJ

    更新日期:2017-08-01 00:00:00

  • Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture.

    abstract::Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables in...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.260400609

    authors: Shah NN,Park S,Taylor RT,Droege MW

    更新日期:1992-09-01 00:00:00

  • Removal of toluene in a vapor-phase bioreactor containing a strain of the dimorphic black yeast Exophiala lecanii-corni.

    abstract::Stricter regulations on volatile organic compounds and hazardous air pollutants have increased the demand for abatement technologies. Biofiltration, a process in which contaminated air is passed through a biologically active bed, can be used to remove these pollutants from air streams. In this study, a fungal vapor-ph...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.10066

    authors: Woertz JR,Kinney KA,McIntosh ND,Szaniszlo PJ

    更新日期:2001-12-05 00:00:00

  • Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production.

    abstract::Chinese hamster ovary (CHO) cells are the predominant host cell line for the production of biopharmaceuticals, a growing industry currently worth more than $188 billion USD in global sales. CHO cells undergo programmed cell death (apoptosis) following different stresses encountered in cell culture, such as substrate l...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章,评审

    doi:10.1002/bit.27269

    authors: Henry MN,MacDonald MA,Orellana CA,Gray PP,Gillard M,Baker K,Nielsen LK,Marcellin E,Mahler S,Martínez VS

    更新日期:2020-04-01 00:00:00

  • A non-chromatographic method for the removal of endotoxins from bacteriophages.

    abstract::The Ff filamentous bacteriophages show potential as a new class of therapeutics, displaying utility in materials science as well as pharmaceutical applications. These phages are produced by the infection of E. coli, a Gram-negative bacterium which unavoidably sheds endotoxins into the extracellular space during growth...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.25571

    authors: Branston SD,Wright J,Keshavarz-Moore E

    更新日期:2015-08-01 00:00:00

  • Photo inactivation of virus particles in microfluidic capillary systems.

    abstract::It has long been established that UVC light is a very effective method for inactivating pathogens in a fluid, yet the application of UVC irradiation to modern biotechnological processes is limited by the intrinsic short penetration distance of UVC light in optically dense protein solutions. This experimental and numer...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.25912

    authors: Ren Y,Crump CM,Mackley MM,Li Puma G,Reis NM

    更新日期:2016-07-01 00:00:00

  • Model-supported optimization of phototrophic growth in a stirred-tank photobioreactor.

    abstract::A fuzzy-logic light attenuation model was developed and validated for a stirred-tank photobioreactor. Based on this model, local light intensities were used to calculate local specific growth rates of the cyanobacteria Synechococcus PCC 7942. The light regime for maximization of biomass space-time yield in a batch pro...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.21086

    authors: Franco-Lara E,Havel J,Peterat F,Weuster-Botz D

    更新日期:2006-12-20 00:00:00

  • Multivariable control of alcohol concentrations in the production of polyhydroxyalkanoates (PHAs) by Paracoccus denitrificans.

    abstract::A novel multivariable control strategy is developed for alcohol (ethanol and n-pentanol) concentrations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(HB-co-HV), a biodegradable polymer by Paracoccus denitrificans ATCC 1774. This controller, which is developed to control the mole fraction of P(HB...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章

    doi:10.1002/bit.1101

    authors: Chanprateep S,Abe N,Shimizu H,Yamane T,Shioya S

    更新日期:2001-07-20 00:00:00