Abstract:
:As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000. In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R(2)≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli). Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.
journal_name
J Biotechnoljournal_title
Journal of biotechnologyauthors
Broger T,Odermatt RP,Huber P,Sonnleitner Bdoi
10.1016/j.jbiotec.2011.05.003subject
Has Abstractpub_date
2011-07-20 00:00:00pages
240-7issue
4eissn
0168-1656issn
1873-4863pii
S0168-1656(11)00234-3journal_volume
154pub_type
杂志文章abstract::Nontypeable Haemophilus influenzae (NTHi) is a gram-negative coccobacillus that is one of the bacteria that form the commensal flora of the upper respiratory tract in humans. This bacterium is an important human pathogen causing a broad spectrum of disease in both adults and children, including invasive and localised ...
journal_title:Journal of biotechnology
pub_type: 杂志文章,评审
doi:10.1016/s0168-1656(99)00113-3
更新日期:1999-08-20 00:00:00
abstract::Aspergillus sojae, which is believed to be a domesticated strain of Aspergillus parasiticus, contains all of the aflatoxin biosynthetic genes but is unable to produce aflatoxins and is generally recognized as safe (GRAS) for producing fermented foods. In A. parasiticus both aflR, the aflatoxin pathway-specific regulat...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2003.10.012
更新日期:2004-02-05 00:00:00
abstract::The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well adapted to each of these substrates was evaluated. Sludge fed with acetate (A), produced a homopolymer of hydroxybutyrate (HB), whereas sludge fed with propionate (P) produced a copolymer of HB and HV (hydoxyvalerate). Switc...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2005.09.006
更新日期:2006-03-23 00:00:00
abstract::Recombinant antibody fragments belong to the promising class of biopharmaceuticals with high potential for future therapeutic applications. However, due to their small size they are rapidly cleared from circulation. Binding to serum proteins can be an effective approach to improve pharmacokinetic properties of short h...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2017.05.017
更新日期:2017-07-10 00:00:00
abstract::A new, chemically defined medium providing dispersed growth and high biomass formation and a method for quantitative extraction of intracellular metabolites was used to investigate the cellular response of Streptomyces coelicolor A3(2) during growth and upon changes in nutrient utilization. Fast changes of the glucose...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/s0168-1656(99)00215-1
更新日期:2000-02-17 00:00:00
abstract::A rapid quantitative analysis method for murine endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC) gene expression levels was established using a real-time polymerase chain reaction (PCR). We designed primer pairs and TaqMan probes specific for murine prepro-ET-1 (PPET-1) and prepro-VIC (PPVIC) genes, base...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/s0168-1656(00)00342-4
更新日期:2001-11-30 00:00:00
abstract::Physiologically relevant in vitro models can serve as biological analytical platforms for testing novel treatments and drug delivery systems. We describe the first steps in the development of a 3D human brain tumour co-culture model that includes the interplay between normal and tumour tissue along with nutrient gradi...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2015.01.002
更新日期:2015-07-10 00:00:00
abstract::Fermented Monascus pigments have been utilized as traditional Chinese medicine and food colorant for thousands of years. Under the limited nitrogen concentration and/or low initial pH 2.5 conditions, it was observed that production of intracellular pigments and accumulation of microbial lipids (high content reaching t...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2015.08.019
更新日期:2015-10-20 00:00:00
abstract::A simple and accurate analysis system for the estimation of recombination efficiency in vivo using fluorescence-activated cell sorting (FACS) was designed and was subsequently used to compare the efficiency of recombination related to different spacer mutants. F(3) and F(5) mutant sequences were used for Flpe-mediated...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2006.07.027
更新日期:2007-01-10 00:00:00
abstract::The first complete genome sequence of Bacillus glycinifermentans B-27 was determined by SMRT sequencing generating a genome sequence with a total length of 4,607,442 bases. Based on this sequence 4738 protein-coding sequences were predicted and used to identify gene clusters that are related to the production of secon...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2017.04.021
更新日期:2017-09-10 00:00:00
abstract::A novel expression vector for yeast has been constructed from the regulatory elements present in the polI promoter and the enhancer/termination region (E/T) of rDNA. Under some conditions, this promoter/vector combination produces small RNAs such as the hammerhead RNA sequence at levels comparable to polII- and polIII...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/s0168-1656(97)00078-3
更新日期:1997-07-23 00:00:00
abstract::The use of enzymes in organic solvents offers a great opportunity for the highly selective synthesis of complex organic compounds. In this study we investigate the POXylation of several enzymes with different polyoxazolines ranging from the hydrophilic poly(2-methyl-oxazoline) (PMOx) to the hydrophobic poly(2-heptyl-o...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2014.03.035
更新日期:2014-07-10 00:00:00
abstract::Transgenic hairy root (HR) systems constitute an interesting alternative to improve the efficiency of phytoremediation process. Since peroxidases (Px) have been associated with phenolic compounds removal, in the present work, transgenic tobacco HR, which expressed basic Px genes from tomato (tpx1 and tpx2), were estab...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2008.11.008
更新日期:2009-02-23 00:00:00
abstract::Dual nutrient limited growth, the control of the cell growth rate (kinetic aspect) or the restriction of the amount of biomass (stoichiometric aspect) by two nutrients at the same time, is a relatively unknown ability of the microorganisms and consequently, still not mentioned in textbooks to date. Nevertheless, multi...
journal_title:Journal of biotechnology
pub_type: 杂志文章,评审
doi:10.1016/j.jbiotec.2004.03.030
更新日期:2004-09-30 00:00:00
abstract::It is quite important to understand the basic principle embedded in the main metabolism for the interpretation of the fermentation data. For this, it may be useful to understand the regulation mechanism based on systems biology approach. In the present study, we considered the perturbation analysis together with compu...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2013.06.023
更新日期:2013-10-20 00:00:00
abstract::The study describes extraction of extracellular polymeric substances (EPS) from sewage sludge by applying enzymes and enzymes combined with sodium tripolyphosphate (STPP). Additionally, a systematic study of two non-enzymatic extraction agents is described. The assessment of the released products is made by colorimetr...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2007.04.005
更新日期:2007-06-30 00:00:00
abstract::The catalytic efficiency and pH optimum of Trichoderma reesei endo-beta-1,4-glucanase II were improved by protein engineering. We subjected residue 342 to saturation mutagenesis, and further changed the enzyme by random mutagenesis and two rounds of DNA shuffling. Enzyme variants were purified and characterized. Varia...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2008.03.016
更新日期:2008-06-01 00:00:00
abstract::Dilute-acid lignocellulosic hydrolyzate was successfully fermented to ethanol by encapsulated Saccharomyces cerevisiae at dilution rates up to 0.5h(-1). The hydrolyzate was so toxic that freely suspended yeast cells could ferment it continuously just up to dilution rate 0.1h(-1), where the cells lost 75% of their viab...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2006.03.013
更新日期:2006-09-18 00:00:00
abstract::Secreted recombinant proteins can aggregate during cell culture. We studied a poorly-behaved bispecific scaffold that increasingly aggregated (up to 62% high molecular weight species, HMW) as a function of culture time in a fed-batch and intensified cell culture processes. We identified that protein aggregates increas...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2019.08.001
更新日期:2019-10-10 00:00:00
abstract::In this work, a proof of concept elastin-like polypeptide-Z domain fusion (ELP-Z) based monoclonal antibody (mAb) affinity precipitation process is developed using scaled-down filtration techniques. Tangential flow filtration (TFF) is examined for the recovery of ELP-Z-mAb precipitates formed during the mAb binding st...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2014.09.020
更新日期:2014-12-20 00:00:00
abstract::A new current amplified immunosensor for the determination of carcinoembryonic antigen (CEA) was demonstrated in this work. The electrode architecture was fabricated by positively charged toluidine blue (TB) coated on negatively charged poly-sulfanilic acid (PSAA) modified glassy carbon electrode (GCE) surface through...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2005.11.023
更新日期:2006-05-29 00:00:00
abstract::Aptamers are single-stranded DNA or RNA oligonucleotides, which have been emerging as recognition elements in disease diagnostics and food control, including the detection of bacterial toxins. In this study, we employed the semi-automated just in time-selection to identify aptamers that bind to cholera toxin (CT) with...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2018.01.012
更新日期:2018-03-10 00:00:00
abstract::Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with β-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product dis...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2015.06.394
更新日期:2015-09-10 00:00:00
abstract::The secreted pore-forming toxin listeriolysin O (LLO) is an essential virulence factor that allows the food-borne bacterial pathogen Listeria monocytogenes to escape from the phagocytic vacuole and reach the host cytosol. This protein belongs to the group of cholesterol-binding sulfhydryl-activated toxins, expressed b...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2003.10.023
更新日期:2004-04-08 00:00:00
abstract::We introduce an easy, fast and effective method to analyze the influence of genetically modified (GM) plants on soil and model organisms in the laboratory to substitute laborious and time consuming field trials. For the studies described here we focused on two GM plants of the so-called 3rd generation: GM plants produ...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2017.07.012
更新日期:2017-09-10 00:00:00
abstract::Structural composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was recently analyzed by means of a metagenome sequencing approach. To determine the transcriptionally active part of the same biogas community and to identify key ...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2012.01.020
更新日期:2012-04-30 00:00:00
abstract::The polyhydroxyalkanoate (PHA) producing capability of four bacterial strains isolated from Antarctica was reported in a previous study. This study analyzed the PHA synthase genes and the PHA-associated gene clusters from the two antarctic Pseudomonas isolates (UMAB-08 and UMAB-40) and the two antarctic Janthinobacter...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2020.03.006
更新日期:2020-04-10 00:00:00
abstract::Bacillus velezensis G341 can suppress plant pathogens by producing antagonistic active compounds including bacillomycin D, fengycin, and (oxy) difficidin. The complete genome sequence of this bacterium was characterized by one circular chromosome of 4,009,746bp with 3953 open reading frames. The genome contained 36 ps...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2015.07.005
更新日期:2015-10-10 00:00:00
abstract::As the important chiral building block of levetiracetam, the synthesis of (S)-2-aminobutanamide has attracted a great deal of attention. The d-aminopeptidase catalyzed kinetic resolution of 2-aminobutanamide was demonstrated as an effective strategy for (S)-2-aminobutanamide production. In this study, a novel d-aminop...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2017.12.003
更新日期:2018-01-20 00:00:00
abstract::Porcine circovirus Cap protein production by P. pastoris with strong AOX promoter suffered with the problems with traditional pure methanol induction: (1) inefficient methanol metabolism; (2) extensive oxygen supply load; (3) difficulty in stable DO control; (4) low protein titer. In this study, based on the differenc...
journal_title:Journal of biotechnology
pub_type: 杂志文章
doi:10.1016/j.jbiotec.2014.02.018
更新日期:2014-05-10 00:00:00