scholarly journals Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor

2014 ◽  
Vol 7 (1) ◽  
Author(s):  
Ariane Leites Larentis ◽  
Júlia Fabiana Monteiro Quintal Nicolau ◽  
Gabriela dos Santos Esteves ◽  
Daniel Tait Vareschini ◽  
Fernanda Vicente Ribeiro de Almeida ◽  
...  
Keyword(s):  
Cell Growth ◽  
E Coli ◽  
Iptg Concentration ◽  
Induction Temperature

scholarly journals Staphylococcus aureus MazF Specifically Cleaves a Pentad Sequence, UACAU, Which Is Unusually Abundant in the mRNA for Pathogenic Adhesive Factor SraP

2009 ◽  
Vol 191 (10) ◽  
pp. 3248-3255 ◽  
Author(s):  
Ling Zhu ◽  
Koichi Inoue ◽  
Satoshi Yoshizumi ◽  
Hiroshi Kobayashi ◽  
Yonglong Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Cell Growth ◽  
Bioinformatics Analysis ◽  
Human Tissues ◽  
Regulatory Relationship ◽  
E Coli ◽  
Pathogenic Factors ◽  
Inhibit Cell Growth ◽  
Adhesive Factor

ABSTRACT Escherichia coli mRNA interferases, such as MazF and ChpBK, are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems present in its genome. A MazF homologue in Staphylococcus aureus (MazFSa) has been shown to inhibit cell growth when induced in E. coli. Here, we determined the cleavage site for MazFSa with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. MazFSa specifically cleaves the RNA at a pentad sequence, U↓ACAU. Bioinformatics analysis revealed that this pentad sequence is significantly abundant in several genes, including the sraP gene in the S. aureus N315 strain. This gene encodes a serine-rich protein, which is known to play an important role in adhesion of the pathogen to human tissues and thus in endovascular infection. We demonstrated that the sraP mRNA became extremely unstable in comparison with the ompA mRNA only when MazFSa was induced in E. coli. Further bioinformatics analysis indicated that the pentad sequence is also significantly abundant in the mRNAs for all the pathogenic factors in S. aureus. This observation suggests a possible regulatory relationship between the MazEFSa TA module and the pathogenicity in S. aureus.

scholarly journals Microbial Synthesis of Non-Natural Anthraquinone Glucosides Displaying Superior Antiproliferative Properties

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2171 ◽  
Author(s):  
Trang Nguyen ◽  
Ramesh Pandey ◽  
Prakash Parajuli ◽  
Jang Han ◽  
Hye Jung ◽  
...  
Keyword(s):  
Cell Growth ◽  
Biological Activities ◽  
Uterine Cervical Cancer ◽  
Cell Growth Inhibition ◽  
Microbial Synthesis ◽  
E Coli ◽  
Anticancer Effects ◽  
Naturally Occurring ◽  
Spectroscopic Analyses ◽  
Proliferative Assay

Anthraquinones, naturally occurring bioactive compounds, have been reported to exhibit various biological activities, including anti-inflammatory, antiviral, antimicrobial, and anticancer effects. In this study, we biotransformed three selected anthraquinones into their novel O-glucoside derivatives, expressing a versatile glycosyltransferase (YjiC) from Bacillus licheniformis DSM 13 in Escherichia coli. Anthraflavic acid, alizarin, and 2-amino-3-hydroxyanthraquinone were exogenously fed to recombinant E. coli as substrate for biotransformation. The products anthraflavic acid-O-glucoside, alizarin 2-O-β-d-glucoside, and 2-amino-3-O-glucosyl anthraquinone produced in the culture broths were characterized by various chromatographic and spectroscopic analyses. The comparative anti-proliferative assay against various cancer cells (gastric cancer-AGS, uterine cervical cancer-HeLa, and liver cancer-HepG2) were remarkable, since the synthesized glucoside compounds showed more than 60% of cell growth inhibition at concentrations ranging from ~50 μM to 100 μM. Importantly, one of the synthesized glucoside derivatives, alizarin 2-O-glucoside inhibited more than 90% of cell growth in all the cancer cell lines tested.

scholarly journals Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli

2020 ◽  
Vol 9 (11) ◽  
pp. 3052-3066
Author(s):  
Meritxell Galindo Casas ◽  
Patrick Stargardt ◽  
Juergen Mairhofer ◽  
Birgit Wiltschi
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Protein Production ◽  
Noncanonical Amino Acids ◽  
Site Specific ◽  
E Coli

scholarly journals In Vivo Modulation of a DnaJ Homolog, CbpA, by CbpM

2007 ◽  
Vol 189 (9) ◽  
pp. 3635-3638 ◽  
Author(s):  
Matthew R. Chenoweth ◽  
Nancy Trun ◽  
Sue Wickner
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Stationary Phase ◽  
Specific Inhibitor ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
E Coli ◽  
Hsp70 Chaperone

ABSTRACT CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

Kinetics and modelling of batch fermentation for the production of organic solvent tolerant and thermostable lipase by recombinant E. coli / Organik çözücü toleranslı ve ısıya dayanıklı rekombinan E. coli lipaz üretiminin kinetiği ve grup fermentasyonu modellemesi

2015 ◽  
Vol 40 (4) ◽  
Author(s):  
Arbakariya Bin Ariff ◽  
Rubina Nelofer ◽  
Raja Nor Zaliha Raja Abdul Rahman ◽  
Mahiran Basri
Keyword(s):  
Cell Growth ◽  
Shake Flask ◽  
Lipase Production ◽  
Stirred Tank ◽  
Thermostable Lipase ◽  
Stirred Tank Bioreactor ◽  
Monod Equation ◽  
E Coli ◽  
Organic Solvent Tolerant ◽  
Solvent Tolerant

AbstractObjective: Kinetics of organic solvent tolerant and thermostable lipase production by recombinant E. coli in shake flask level and 2 L stirred tank bioreactor level was studied to observe the variations of important kinetic parameters at two different levels of bioprocess.Methods: Unstructured models based on Monod equation for growth and Luedeking-Piret equation for lipase production and glucose consumption were used to predict cell growth, lipase production and glucose utilization. The shake flask fermentation experiments were carried out at different initial glucose and yeast extract concentrations using recombinant bacterial strain E. coli BL21. Lipase production was also carried out using 2L stirred tank bioreactor for comparison.Results: In all cases, the data fitted well to the proposed models. The highest growth and lipase activity were obtained at 25 g/L glucose and 25 g/L yeast extract. Cell growth (6.42 g/L) and lipase production (65.32 IU/mL) in 2 L stirred tank bioreactor was comparable to those obtained in shake flask fermentations. The calculated value of growth associated constant (9.874 IU/g/h) was much higher than that of non-growth associated constant (0.022 IU/g/h) in bioreactor as well as in shake flasks. The values of maximum specific growth rate (μm) and glucose saturation constant (KS) for shake flask fermentations, calculated from Monod equation, were 0.476 h-1 and 5.237 g/L respectively.Conclusion: From the modelling exercise, it was concluded that the lipase production is dominantly growth associated process. The kinetic parameter values for fermentations in shake flask and 2L stirred tank bioreactor were comparable, indicating that the bioprocess could be transferred into larger scale.

scholarly journals Possible epigenetic influence on cellular differentiation and lineage segregation in Escherichia coli

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Cell Growth ◽  
Single Cell ◽  
Cell Fate ◽  
Cellular Differentiation ◽  
Environmental Stressors ◽  
Epigenetic Markers ◽  
E Coli ◽  
Growth Chambers

Epigenetics provides the critical connection between environmental influence and gene expression, where environmental stressors could modulate expression of specific genes in particular scenarios using molecular markers etched at the genome level. Hence, epigenetics likely play important roles in potentiating the development of specific lineages, cell fate or cellular differentiation. For example, when specific environmental stressor is present, epigenetic markers in the genome receive a signal for either activating or deactivating expression of particular sets of genes, which may be linked to the developmental trajectory of the organism. Using Escherichia coli as model organism, a possible study may investigate the role of epigenetics in influencing cellular differentiation of the bacterium. Specifically, a single E. coli cell would be propagated into a consortium of 12 or more bacterial cells in a microfluidics growth chamber. Genetic material extracted would be sent for single cell genomics, transcriptomics, and chromatin immunoprecipitation sequencing (ChIP-seq). After profiling, the residual population would be diverted by microchannels to 6 different cell growth chambers, where they would be cultivated under identical conditions for understanding possible triggers to cell differentiation. At suitable time points of 2, 4, 6, 8, 10, 12 hours, single cell would be extracted from each growth chamber for profiling single cell genomics, transcriptomics, and epigenetics markers. Optical and confocal laser scanning microscopy would provide readout of cell morphologies. Comparison of the readout between the original clonal population and those of the different growth chambers may provide important points for correlating epigenetic markers with gene expression and phenotypic readout in cell lineage, fate and differentiation. In subsequent experiments, different environmental stressors such as pH, imbalance nutrient composition between carbon and nitrogen, nanoparticles or heavy metals, could be used as triggers for specific cell growth response guided by epigenetic programmes embedded within the epigenome of the bacterium. Collectively, epigenetics hold influence for cellular differentiation in view of specific environmental stressors, where epigenetic markers on the genome communicate specific environmental factor's effect on the organism through altering expression of particular sets of genes, that result in different cell fate, lineage and differentiation. Using modern single cell techniques at the genomics, transcriptomics and epigenomics level, the study hopes to elucidate epigenetic potentiators of cellular differentiation in E. coli with and without environmental stressors such as nutrient deprivation, pH and toxic metals.

scholarly journals Reproducing bench-scale cell growth and productivity

2018 ◽  
Author(s):  
Ariel Hecht ◽  
James Filliben ◽  
Sarah A. Munro ◽  
Marc Salit
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Small Molecule ◽  
Fluorescent Protein ◽  
Fractional Factorial ◽  
Red Fluorescent Protein ◽  
Factorial Experiments ◽  
Minimum Information ◽  
E Coli ◽  
Product Titer

Reproducing, exchanging, comparing, and building on each other’s work is foundational to technology advances.1Advancing biotechnology calls for reliable reuse of engineered strains.2Reliable reuse of engineered strains requires reproducible growth and productivity. To demonstrate reproducibility for biotechnology, we identified the experimental factors that have the greatest effect on the growth and productivity of our engineered strains.3–6We present a draft of a Minimum Information Standard for Engineered Organism Experiments (MIEO) based on this method. We evaluated the effect of 22 factors onEscherichia coli(E. coli) engineered to produce the small molecule lycopene, and 18 factors onE. coliengineered to produce red fluorescent protein (RFP). Container geometry and shaking had the greatest effect on product titer and yield. We reproduced our results under two different conditions of reproducibility:7conditions of use (different fractional factorial experiments), and time (48 biological replicates performed on 12 different days over four months).

scholarly journals Production of soluble regulatory hydrogenase from Ralstonia eutropha in Escherichia coli using a fed-batch-based autoinduction system

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Qin Fan ◽  
Peter Neubauer ◽  
Matthias Gimpel
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Protein Production ◽  
Ralstonia Eutropha ◽  
Process Time ◽  
Synthesis Rate ◽  
Heterologous Production ◽  
Fed Batch ◽  
Iptg Induction ◽  
E Coli

Abstract Background Autoinduction systems can regulate protein production in Escherichia coli without the need to monitor cell growth or add inducer at the proper time following culture growth. Compared to classical IPTG induction, autoinduction provides a simple and fast way to obtain high protein yields. In the present study, we report on the optimization process for the enhanced heterologous production of the Ralstonia eutropha regulatory hydrogenase (RH) in E. coli using autoinduction. These autoinduction methods were combined with the EnPresso B fed-batch like growth system, which applies slow in situ enzymatic glucose release from a polymer to control cell growth and protein synthesis rate. Results We were able to produce 125 mg L−1 RH corresponding to a productivity averaged over the whole process time of 3 mg (L h)−1 in shake flasks using classic single-shot IPTG induction. IPTG autoinduction resulted in a comparable volumetric RH yield of 112 mg L−1 and due to the shorter overall process time in a 1.6-fold higher productivity of 5 mg (L h)−1. In contrast, lactose autoinduction increased the volumetric yield more than 2.5-fold and the space time yield fourfold reaching 280 mg L−1 and 11.5 mg (L h)−1, respectively. Furthermore, repeated addition of booster increased RH production to 370 mg L−1, which to our knowledge is the highest RH concentration produced in E. coli to date. Conclusions The findings of this study confirm the general feasibility of the developed fed-batch based autoinduction system and provide an alternative to conventional induction systems for efficient recombinant protein production. We believe that the fed-batch based autoinduction system developed herein will favor the heterologous production of larger quantities of difficult-to-express complex enzymes to enable economical production of these kinds of proteins.

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