Abstract:
: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.
journal_name
Biotechnol Bioengjournal_title
Biotechnology and bioengineeringauthors
Du L,Gao R,Forster ACdoi
10.1002/bit.22491subject
Has Abstractpub_date
2009-12-15 00:00:00pages
1189-96issue
6eissn
0006-3592issn
1097-0290journal_volume
104pub_type
杂志文章abstract::The discovery of denitrifying anaerobic methane oxidation (DAMO) processes enables the complete nitrogen removal from wastewater by utilizing the methane produced on site from anaerobic digesters. This model-based study investigated the mechanisms and operational window for efficient nitrogen removal by coupling nitri...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.25866
更新日期:2016-05-01 00:00:00
abstract::As the therapeutic monoclonal antibody (mAb) market continues to grow, optimizing production processes is becoming more critical in improving efficiencies and reducing cost-of-goods in large-scale production. With the recent trends of increasing cell culture titers from upstream process improvements, downstream capaci...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.24377
更新日期:2012-04-01 00:00:00
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
更新日期:2020-03-01 00:00:00
abstract::At the most fundamental level, saccharification occurs when cell wall degrading enzymes (CWDEs) diffuse, bind to and react on readily accessible cellulose fibrils. Thus, the study of the diffusive behavior of solutes into and out of cellulosic substrates is important for understanding how biomass pore size distributio...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.24958
更新日期:2013-11-01 00:00:00
abstract::Innovation in monoclonal antibody (mAb) production continues to be driven by cell engineering strategies to increase yield and improve product quality. In a previous study, to investigate the effectiveness of transporter overexpression strategies, we prepared a taurine transporter-overexpressing host cell line (DXB11/...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.24881
更新日期:2013-08-01 00:00:00
abstract::The lignin peroxidases (LIP) and manganese peroxidases (MNP) of Phanerochaete chrysosporium catalyze a wide range of lignin depolymerization reactions with lignin models and synthetic lignins in solution. However, their ability to degrade insoluble natural lignin in aqueous media has not been demonstrated. Insoluble i...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/(sici)1097-0290(19980320)57:6<704::aid-bit
更新日期:1998-03-20 00:00:00
abstract::Invertase from Candida utilis was immobilized on porous cellulose beads by an ionic-quanidino bond. The immobilized invertase showed optimum activity between pH 4.0 and 5.4, while the free enzyme had a sharp optimum at pH 4.1. Both temperature profiles were fairly similar up to 55 degrees C. However, above this temper...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.260190307
更新日期:1977-03-01 00:00:00
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
更新日期:2015-08-01 00:00:00
abstract::A microarray presenting glycerol nanodroplets of fluorogenic peptide substrates was used as a biosensor for the detection of multiple enzyme activities within human plasma. Using 10 different plasma proteases (kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa, factor Xa, thrombin, activated protein C, uPA a...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.20927
更新日期:2006-08-20 00:00:00
abstract::An immobilized enzyme (pancreatic ribonuclease bound to porous titania) was investigated for the degradation of purified yeast ribonucleic acid as a substrate. The immobilized enzyme is active and stable in the pH range 4--8. Dependence of enzymatic activity on ionic strength, pH, temperature, fluid flow rate, and sub...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.260210910
更新日期:1979-09-01 00:00:00
abstract::An extractive acetonobutylic fermentation process is developed by integrating bioproduction, Ultrafiltration, and distillation, providing simultaneous retention of biomass, selective removal of inhibitors from the permeate, as well as separation and purification of acetone-butanol-ethanol solvents. Successive batch fe...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.260350903
更新日期:1990-04-15 00:00:00
abstract::In this paper an extensive batch experiment of endogenous process behavior in an aerobic biodegradation process is presented. From these experimental data, comprising measurements of MLVSS (mixed liquor volatile suspended solids) and respiration rate, in a first step the states and unknown parameters in a four-compart...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:
更新日期:2000-05-20 00:00:00
abstract::The insect larvae/baculovirus protein production process was dramatically simplified by expressing fusion proteins containing green fluorescent protein (GFP) and the product-of-interest. In this case, human interleukin-2 (hIL-2) and chloramphenicol acetyl-transferase (CAT) were model products. Specifically, our fusion...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/(sici)1097-0290(19991105)65:3<316::aid-bit
更新日期:1999-11-05 00:00:00
abstract::Engineered strains of Saccharomyces cerevisiae are used for industrial production of succinic acid. Optimal process conditions for dicarboxylic-acid yield and recovery include slow growth, low pH, and high CO2 . To quantify and understand how these process parameters affect yeast physiology, this study investigates in...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.27210
更新日期:2020-03-01 00:00:00
abstract::In this work the question was addressed if in nitrite-oxidizing activated sludge systems the environmental competition between Nitrobacter spp. and Nitrospira spp., which only recently has been discovered to play a role in these systems, is affected by the nitrite concentrations. Two parallel chemostats were inoculate...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.21004
更新日期:2006-09-05 00:00:00
abstract::A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to high ethanol concentrations (10%, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-ba...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.22181
更新日期:2009-04-01 00:00:00
abstract::Reactive oxygen species (ROS), encompassing all oxygen radical or non-radical oxidizing agents, play key roles in disease progression. Controlled delivery of antioxidants is therapeutically relevant in such oxidant-stressed environments. Encapsulating small hydrophilic molecules into hydrophobic polymer microparticles...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.26443
更新日期:2018-01-01 00:00:00
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
doi:10.1002/bit.22139
更新日期:2009-03-01 00:00:00
abstract::There are a series of examples in which phenols appear as contaminants in process streams and their selective removal is required for waste minimization. For the selective removal of a phenol from a mixture, we are exploiting the substrate specificity of the enzyme tyrosinase to convert phenols into reactive o-quinone...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/(SICI)1097-0290(19960705)51:1<79::AID-BIT9
更新日期:1996-07-05 00:00:00
abstract::An in-situ, mid-infrared sensor was used to monitor the major analyte concentrations involved in the cultivation of Gluconacetobacter xylinus and the production of gluconacetan, a food-grade exopolysaccharide. To predict the analyte concentrations, three different sets of standard spectra were used to develop calibrat...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.10618
更新日期:2003-06-20 00:00:00
abstract::The placement of the reference electrode (RE) in various bioelectrochemical systems is often varied to accommodate different reactor configurations. While the effect of the RE placement is well understood from a strictly electrochemistry perspective, there are impacts on exoelectrogenic biofilms in engineered systems ...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.25253
更新日期:2014-10-01 00:00:00
abstract::The production of volatile industrial chemicals utilizing metabolically engineered extreme thermophiles offers the potential for processes with simultaneous fermentation and product separation. An excellent target chemical for such a process is acetone (Tb = 56°C), ideally produced from lignocellulosic biomass. Caldi...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.27529
更新日期:2020-08-08 00:00:00
abstract::The cellulase production from immobilized Trichoderma reesei composites prepared by radiation polymerization at low temperature was studied. The production of cellulase from the cells irradiated by radiation was slightly retarded at the initial stage of the culture, but was immediately recovered. The production of cel...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.260260105
更新日期:1984-01-01 00:00:00
abstract::Mathematical models to describe transcription (Arnold et al. (2001); Biotech Bioeng 72:548-561) and translation (Mehra et al. (2003); Biotech Bioeng 84:822-841) in bacteria are modified in order to improve reaction kinetics and to include the number of polymerase molecules that are active on the DNA, as well as to inc...
journal_title:Biotechnology and bioengineering
pub_type: 评论,信件
doi:10.1002/bit.21065
更新日期:2007-03-01 00:00:00
abstract::Cellulase (Cellulosin AC-8) was immobilized on poly-L-glutamic acid. This immobilized cellulase (IC) is water soluble in the neutral and alkaline solutions, where IC has the activity, while IC can be made insoluble by lowering the pH so that it can be recovered from the reaction mixture with its activity. The optimum ...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.260290203
更新日期:1987-02-01 00:00:00
abstract::Retroviral-derived biopharmaceuticals (RV) target numerous therapeutic applications, from gene therapy to virus-like particle (rVLP)-based vaccines. During particle formation, beside the pseudotyped envelope proteins, RV can incorporate proteins derived from the virus producer cells (VPC). This may be detrimental by r...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.23231
更新日期:2011-11-01 00:00:00
abstract::Coastal bermudagrass was pretreated by a low-temperature ammonia fiber explosion (AFEX) process, which soaked the grass in liquid ammonia and then explosively released the pressure. Saccharifying enzymes were systematically applied to the AFEX-treated grass corresponding to low, medium, and high loadings of cellulase/...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.260440914
更新日期:1994-11-05 00:00:00
abstract::Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure-cycled bioreactor for animal cell cultures, described previou...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.23289
更新日期:2012-01-01 00:00:00
abstract::The aim of this study was to determine the feasibility of using high pressure CO2 for sterilization of Ginseng powder, as an alternative method to conventional techniques such as gamma-irradiation and ethylene oxide. The Ginseng sample used in this study was originally contaminated with fungi and 5 x 10(7) bacteria/g ...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.22059
更新日期:2009-02-01 00:00:00
abstract::The reconstruction of functioning human tissues ex vivo is becoming an important part of biotechnology. There are compelling scientific, clinical, and biotechnological reasons for fully or partially reconstituting human tissues such as skin, bone marrow, and liver ex vivo. In particular, bone marrow is a tissue of muc...
journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
doi:10.1002/bit.260420802
更新日期:1993-10-01 00:00:00