Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase.


:Engineering protein expression in vitro or in vivo is usually straightforward for single genes, but remains challenging for multiple genes because of the requirement of coordinated control. RNA and protein overexpression strategies often exploit T7 RNA polymerase and its natural TPhi Class I terminator. However, this terminator's inefficiency and large size (100 bp) are problematic for multigene construction and expression. Here, we measure the effects of tandem copies of a small (18 bp) Class II T7 terminator from vesicular stomatitis virus on transcription in vitro and on translation in vitro and in vivo. We first test monomeric and dimeric gene constructs, then attempt extension to pentameric gene constructs. "BioBrick" versions of a pET vector and translation factor genes were constructed to facilitate cloning, and His-tags were incorporated to allow copurification of all protein products for relatively unbiased analysis and easy purification. Several results were surprising, including imbalanced expression of the pentameric constructs in vivo, illustrating the value of synthetic biology for investigating gene expression. However, these problems were solved rationally by changing the orders of the genes and by adding extra promoters to the upstream gene or by moving to a more predictable in vitro translation system. These successes were significant, given our initial unexpected results and that we are unaware of another example of coordinated overexpression of five proteins. Our modular, flexible, rational method should further empower synthetic biologists wishing to overexpress multiple proteins simultaneously.


Biotechnol Bioeng


Du L,Gao R,Forster AC




Has Abstract


2009-12-15 00:00:00












  • The effect of temperature on the continuous ferrous-iron oxidation kinetics of a predominantly Leptospirillum ferrooxidans culture.

    abstract::The ferrous-iron oxidation kinetics of a bacterial culture consisting predominantly of Leptospirillum ferrooxidans were studied in continuous-flow bioreactors. The bacterial culture was fed with a salts solution containing 12 g/L ferrous-iron, at dilution rates ranging from 0.01 to 0.06 l/h, and temperatures ranging f...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Breed AW,Dempers CJ,Searby GE,Gardner MN,Rawlings DE,Hansford GS

    更新日期:1999-10-05 00:00:00

  • Alginate as immobilization material: III. Diffusional properties.

    abstract::The rate of diffusion of serum albumin (MW 6.9 x 10(4) D) out of beads of calcium alginate gels depends upon the concentration and uronic acid composition of the alginate (ManA/GulA ratio), the conditions under which the beads are produced, the pH, and the temperature. The diffusion coefficient decreases with increasi...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Martinsen A,Storrø I,Skjårk-Braek G

    更新日期:1992-01-20 00:00:00

  • A novel aryl acylamidase from Nocardia farcinica hydrolyses polyamide.

    abstract::An alkali stable polyamidase was isolated from a new strain of Nocardia farcinica. The enzyme consists of four subunits with a total molecular weight of 190 kDa. The polyamidase cleaved amide and ester bonds of water insoluble model substrates like adipic acid bishexylamide and bis(benzoyloxyethyl)terephthalate and hy...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Heumann S,Eberl A,Fischer-Colbrie G,Pobeheim H,Kaufmann F,Ribitsch D,Cavaco-Paulo A,Guebitz GM

    更新日期:2009-03-01 00:00:00

  • Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture.

    abstract::Recombinant retroviruses are effective vectors able to integrate transgenes into the target cell's genome to achieve longer-term expression. This study investigates the effect of cell lysis products, a common cell culture by-product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates deri...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Beauchesne PR,Bruce KJ,Bowen BD,Piret JM

    更新日期:2010-04-15 00:00:00

  • Kinetic analysis of cephalosporin biosynthesis in Streptomyces clavuligerus.

    abstract::A kinetic model describing the cephalosporin biosynthesis in Streptomyces clavuligerus was developed. Using previously reported kinetic data of biosynthetic enzymes, we examined the kinetics of cephalosporin production. The predicted time profile of the specific production rate during a batch culture parallels that of...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Malmberg LH,Hu WS

    更新日期:1991-10-20 00:00:00

  • Biosorption of lead and nickel by biomass of marine algae.

    abstract::Screening tests of different marine algae biomas types revealed a high passive biosorptive uptake of lead up to 270 mg Pb/g of biomass in some brown marine algae. Members of the order Fucales perfomed particularly well in this descending sequence: Fucus > Ascophyllum > Sargassum. Although decreasing the swelling of we...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Holan ZR,Volesky B

    更新日期:1994-05-01 00:00:00

  • Chemical biotechnology for the specific oxyfunctionalization of hydrocarbons on a technical scale.

    abstract::Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means. Many enzymatic oxygenations have been described, but few of these have been scaled up to industrial scales, due to ...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Bühler B,Bollhalder I,Hauer B,Witholt B,Schmid A

    更新日期:2003-06-30 00:00:00

  • Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate.

    abstract::The rate of celluose degradation, limited due to the inhibition by cellobiose, can be increased by the hydrolysis of cellobiose to glucose using immobilized beta-glucosidase. Production of beta-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii ...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Jain D,Ghose TK

    更新日期:1984-04-01 00:00:00

  • Cell cycle progression in Escherichia coli B/r affects transcription of certain genes: Implications for synthetic genome design.

    abstract::We propose that transcript levels for some genes are affected by the bacterial cell division cycle and this may be an important factor to consider when designing synthetic bacterial genomes. To test this hypothesis, transcript levels of 58 genes in Escherichia coli B/r A were determined at five times during the cell d...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Echtenkamp PL,Wilson DB,Shuler ML

    更新日期:2009-02-15 00:00:00

  • Novel application of oxygen-transferring membranes to improve anaerobic wastewater treatment.

    abstract::Anaerobic biological wastewater treatment has numerous advantages over conventional aerobic processes; anaerobic biotechnologies, however, still have a reputation for low-quality effluents and operational instabilities. In this study, anaerobic bioreactors were augmented with an oxygen-transferring membrane to improve...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Kappell AS,Semmens MJ,Novak PJ,LaPara TM

    更新日期:2005-02-20 00:00:00

  • Effect of electrical charges and fields on injury and viability of airborne bacteria.

    abstract::In this study, the effects of the electric charges and fields on the viability of airborne microorganisms were investigated. The electric charges of different magnitude and polarity were imparted on airborne microbial cells by a means of induction charging. The airborne microorganisms carrying different electric charg...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Mainelis G,Górny RL,Reponen T,Trunov M,Grinshpun SA,Baron P,Yadav J,Willeke K

    更新日期:2002-07-20 00:00:00

  • The acetone butanol fermentation on glucose and xylose. I. Regulation and kinetics in batch cultures.

    abstract::The kinetics in batch culture of the acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The fastest initial growth and transition from an acid to a solvent metabolism occurs on glucose, with a final 62 g/L glucose conversion. On xylose, an initial sl...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Fond O,Engasser JM,Matta-El-Amouri G,Petitdemange H

    更新日期:1986-02-01 00:00:00

  • A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae.

    abstract::Pathway refactoring serves as an invaluable synthetic biology tool for natural product discovery, characterization, and engineering. However, the complicated and laborious molecular biology techniques largely hinder its application in natural product research, especially in a high-throughput manner. Here we report a p...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Ren H,Hu P,Zhao H

    更新日期:2017-08-01 00:00:00

  • Egr1 and Gas6 facilitate the adaptation of HEK-293 cells to serum-free media by conferring enhanced viability and higher growth rates.

    abstract::Animal-derived serum is an essential media supplement for mammalian cells in cell culture. For a number of reasons including cost, regulatory concerns, lot inconsistency, potential contamination with adventitious agents, and down-stream processing it is desirable to eliminate the use of serum. Existing protocols desig...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Jaluria P,Konstantopoulos K,Betenbaugh M,Shiloach J

    更新日期:2008-04-15 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


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

    更新日期:2015-08-01 00:00:00

  • Enzymatic synthesis of esters using an immobilized lipase.

    abstract::Various esters were synthesized in nearly anhydrous hexane from alcohols and carboxylic acids using a lipase from Candida cylindracea. The enzyme was immobilized on a nylon support and protein loadings as high as 10 mg/g were obtained. The activity of the immobilized enzyme was maximum in a range of temperatures from ...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Carta G,Gainer JL,Benton AH

    更新日期:1991-05-01 00:00:00

  • Recovery of proteins and amino acids from reverse micelles by dehydration with molecular sieves.

    abstract::A new method is presented to precipitate proteins and amino acids from reverse micelles by dehydrating the micelles with molecular sieves. Nearly complete precipitation is demonstrated for alpha-chymotrypsin, cytochromec, and trytophan from 2-ethylhexyl sodium sulfosuccinate (AOT)/isooctane/water reverse micelle solut...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Gupta RB,Han CJ,Johnston KP

    更新日期:1994-09-20 00:00:00

  • A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization.

    abstract::Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approache...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章,评审


    authors: Lima JF,Maia P,T Magalhães B,Cerqueira L,Azevedo NF

    更新日期:2020-10-01 00:00:00

  • Temperature-dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli.

    abstract::Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two-stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Harder BJ,Bettenbrock K,Klamt S

    更新日期:2018-01-01 00:00:00

  • Role of some whey components on mass transfer in ultrafiltration.

    abstract::Ultrafiltration through Carbosep M(4) mineral membrane of protein solutions of decreasing complexity (whey before and after centrifugation or clarification, beta-lactoglobulin) was studied. Mathematical models were used to explain variations in flux with time. Taking into account variations in protein retention and hy...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Taddei C,Daufin G,Aimar P,Sanchez V

    更新日期:1989-06-20 00:00:00

  • The inhibition of the maximum specific growth and fermentation rate of Zymomonas mobilis by ethanol.

    abstract::The inhibition of the maximum specific growth and fermentation rate of Zymomonas mobilis by ethanol was studied in turbidostat cultures at constant and stepwise changed ethanol concentrations. Up to 50 g/L ethanol, the inhibition kinetics can be approximated by a linear relationship between the specific growth rate an...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Jöbses IM,Roels JA

    更新日期:1986-04-01 00:00:00

  • Simple approach to reducing proteolysis during secretory production of human parathyroid hormone in Saccharomyces cerevisiae.

    abstract::A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor alpha pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the p...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Chung BH,Park KS

    更新日期:1998-01-20 00:00:00

  • Thermodynamic analysis of solvent effect on substrate specificity of lyophilized enzymes suspended in organic media.

    abstract::A simple methodology has been successfully employed to explain the solvent dependence of the substrate specificity of enzymes in organic media. This methodology, which does not require the knowledge of the enzyme structure and is thus applicable to lyophilized and other noncrystalline enzyme preparations, predicts tha...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Wescott CR,Klibanov AM

    更新日期:1997-11-05 00:00:00

  • Diffusion of lactate and ammonium in relation to growth of Geotrichum candidum at the surface of solid media.

    abstract::Geotrichum candidum was cultivated at the surface of solid model media containing peptone to simulate the composition of Camembert cheese. The surface growth of G. candidum induced the diffusion of substrates from the core to the rind and the diffusion of produced metabolites from the rind to the core. In the range of...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Aldarf M,Fourcade F,Amrane A,Prigent Y

    更新日期:2004-07-05 00:00:00

  • Coaxial electrohydrodynamic spraying of plasmid DNA/polyethylenimine (PEI) polyplexes for enhanced nonviral gene delivery.

    abstract::DNA/polyethylenimine (PEI) polyplexes are an important class of nonviral vectors. Although the conventional preparation method, bulk mixing, is straightforward, the formation of the DNA/PEI polyplexes is not well controlled. This work explores coaxial electrohydrodynamic spraying (EHDS) as a novel, alternative method ...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Wu Y,Fei Z,Lee LJ,Wyslouzil BE

    更新日期:2010-03-01 00:00:00

  • Expanded bed adsorption/desorption of proteins with Streamline Direct CST I adsorbent.

    abstract::Streamline Direct CST I is a new type of ion exchanger with multi-modal functional groups, specially designed for an expanded bed adsorption (EBA) process, which can capture directly the proteins from the high ionic strength feedstocks with a high binding capacity. In this study, an experimental study is carried out f...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Li P,Xiu G,Mata VG,Grande CA,Rodrigues AE

    更新日期:2006-08-20 00:00:00

  • Bromoperoxidase activity in microplantlet suspension cultures of the macrophytic red alga Ochtodes secundiramea.

    abstract::Bromoperoxidase is an enzyme found in marine macroalgae that catalyzes the bromination of organic substrates. Photosynthetic microplantlet suspension cultures derived from the macrophytic red alga Ochtodes secundiramea were shown to possess bromoperoxidase. The optimum pH for O. secundiramea bromoperoxidase activity i...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Rorrer GL,Tucker MP,Cheney DP,Maliakal S

    更新日期:2001-09-05 00:00:00

  • Population balance modeling of the conidial aggregation of Aspergillus niger.

    abstract::Numerous biotechnological production processes are based on the submerse cultivation of filamentous fungi. Process design, however, is often hampered by the complex growth pattern of these organisms. In the morphologic development of coagulating filamentous fungi, like Aspergillus niger, conidial aggregation is the fi...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Lin PJ,Grimm LH,Wulkow M,Hempel DC,Krull R

    更新日期:2008-02-01 00:00:00

  • Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase.

    abstract::The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutin...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Baptista RP,Cabral JM,Melo EP

    更新日期:2000-12-20 00:00:00

  • Two-stage fermentation with bacteriophage lambda as an expression vector in Escherachia coli.

    abstract::The potential of bacteriophage lambda as an expression vector for a large scale production of cloned-gene proteins was evaluated in batch and continuous bioreactors using a temperature-sensitive mutant in the cl gene, which allows a simple manipulation of temperature as a means to control the phage in the lysogenic or...

    journal_title:Biotechnology and bioengineering

    pub_type: 杂志文章


    authors: Park TH,Seo JH,Lim HC

    更新日期:1991-02-20 00:00:00