scholarly journals Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction in E. coli

2020 ◽  
Author(s):  
Eirik A Moreb ◽  
Zhixia Ye ◽  
John P Efromson ◽  
Jennifer N Hennigan ◽  
Romel Menacho-Melgar ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Heterologous Protein ◽  
Scale Up ◽  
Mineral Salts ◽  
Two Stage ◽  
Robustness Testing ◽  
E Coli ◽  
Media Formulation ◽  
The Impact ◽  
Regulated Promoters

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the robustness testing of a small library of E. coli PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media. Media dependent differences were promoter dependent. 4 out of 16 promoters tested were identified to have tightly controlled expression which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. This subset of PhoB activated promoters, useful for two-stage autoinduction, highlight the impact of the environment on the performance of biological parts, and the importance of robustness testing in synthetic biology.

scholarly journals Microbiologic effectiveness of boiling and safe water storage in South Sulawesi, Indonesia

2011 ◽  
Vol 9 (3) ◽  
pp. 577-585 ◽  
Author(s):  
Samir V. Sodha ◽  
M. Menon ◽  
K. Trivedi ◽  
A. Ati ◽  
M. E. Figueroa ◽  
...  
Keyword(s):  
Water Quality ◽  
Water Samples ◽  
Scale Up ◽  
Treatment Practices ◽  
Rural Districts ◽  
E Coli ◽  
South Sulawesi ◽  
The Government ◽  
The Impact ◽  
Prevalence Ratios

In Indonesia, where diarrhea remains a major cause of mortality among children <5 years, the government promotes boiling of drinking water. We assessed the impact of boiling on water quality in South Sulawesi. We surveyed randomly selected households with at least one child <5 years old in two rural districts and tested source and stored water samples for Escherichia coli contamination. Among 242 households, 96% of source and 51% of stored water samples yielded E. coli. Unboiled water samples, obtained from 15% of households, were more likely to yield E. coli than boiled samples [prevalence ratios (PR) = 2.0, 95% confidence interval (CI) 1.7–2.5]. Water stored in wide-mouthed (PR = 1.4, 95% CI = 1.1–1.8) or uncovered (PR = 1.8, 95% CI = 1.3–2.4) containers, or observed to be touched by the respondent's hands (PR = 1.6, 95% CI = 1.3–2.1) was more likely to yield E. coli. A multivariable model showed that households that did not boil water were more likely to have contaminated stored water than households that did boil water (PR = 1.9, 95% CI = 1.5–2.3). Although this study demonstrated the effectiveness of boiling in reducing contamination, overall impact on water quality was suboptimal. Future studies are needed to identify factors behind the success of boiling water in Indonesia to inform efforts to scale up other effective water treatment practices.

Recombinant Protein Production and Plasmid Stability in Escherichia coli Biofilms

2020 ◽  
Author(s):  
Luciana C. Gomes ◽  
Gabriel A. Monteiro ◽  
Filipe J. Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Heterologous Protein ◽  
Plasmid Stability ◽  
Gene Dosage ◽  
Heterologous Protein Expression ◽  
E Coli ◽  
The Impact

<p><em>Escherichia coli</em> biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.</p> <p>In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from <em>E. coli</em> biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of <em>E. coli</em> JM109(DE3) transformed with a plasmid containing a T7 promoter.</p> <p>It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].</p> <p>It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.</p> <p> </p> <p> </p> <p>References:</p> <p>[1] Gomes, L.C., & Mergulhão, F.J. (2017) Heterologous protein production in <em>Escherichia coli</em> biofilms: A non-conventional form of high cell density cultivation. <em>Process Biochemistry, 57, 1-8</em>. https://doi.org/10.1016/j.procbio.2017.03.018</p> <p>[2] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by <em>Escherichia coli</em> Biofilms. <em>International Journal of Molecular Sciences, 21(2), 576</em>. https://doi.org/10.3390/ijms21020576</p>

scholarly journals Developing synthetic biology for industrial biotechnology applications

2020 ◽  
Vol 48 (1) ◽  
pp. 113-122
Author(s):  
Lionel Clarke ◽  
Richard Kitney
Keyword(s):  
Synthetic Biology ◽  
Scale Up ◽  
Private Investment ◽  
Consumer Products ◽  
Industrial Biotechnology ◽  
Investment Activity ◽  
Ongoing Research ◽  
The Us ◽  
Start Ups ◽  
The Impact

Since the beginning of the 21st Century, synthetic biology has established itself as an effective technological approach to design and engineer biological systems. Whilst research and investment continues to develop the understanding, control and engineering infrastructural platforms necessary to tackle ever more challenging systems — and to increase the precision, robustness, speed and affordability of existing solutions — hundreds of start-up companies, predominantly in the US and UK, are already translating learnings and potential applications into commercially viable tools, services and products. Start-ups and SMEs have been the predominant channel for synthetic biology commercialisation to date, facilitating rapid response to changing societal interests and market pull arising from increasing awareness of health and global sustainability issues. Private investment in start-ups across the US and UK is increasing rapidly and now totals over $12bn. Health-related biotechnology applications have dominated the commercialisation of products to date, but significant opportunities for the production of bio-derived materials and chemicals, including consumer products, are now being developed. Synthetic biology start-ups developing tools and services account for between 10% (in the UK) and ∼25% (in the US) of private investment activity. Around 20% of synthetic biology start-ups address industrial biotechnology targets, but currently, only attract ∼11% private investment. Adopting a more networked approach — linking specialists, infrastructure and ongoing research to de-risk the economic challenges of scale-up and supported by an effective long-term funding strategy — is set to transform the impact of synthetic biology and industrial biotechnology in the bioeconomy.

scholarly journals Media Robustness and Scalability of Phosphate Regulated Promoters Useful for Two-Stage Autoinduction in E. coli

2020 ◽  
Vol 9 (6) ◽  
pp. 1483-1486 ◽  
Author(s):  
Eirik Adim Moreb ◽  
Zhixia Ye ◽  
John P. Efromson ◽  
Jennifer N. Hennigan ◽  
Romel Menacho-Melgar ◽  
...  
Keyword(s):  
Two Stage ◽  
E Coli ◽  
Regulated Promoters

scholarly journals Pathways Analyzer: Design of a Tool for the Synthetic Assembly of Escherichia Coli K-12 MG1655 Bacteria for Biofuel Production

2019 ◽  
Author(s):  
Laura Vasquez ◽  
Ricardo Alvarado ◽  
Allan Orozco
Keyword(s):  
Synthetic Biology ◽  
Metabolic Pathways ◽  
Point Of View ◽  
Biofuel Production ◽  
Design Stage ◽  
Computational Point ◽  
E Coli ◽  
Process Waste ◽  
K 12 ◽  
The Impact

ABSTRACTSummaryDue to the impact of environmental pollution, the importance of producing high quality biofuels and to leverage organic waste that normally would have no use has increased over time. Through synthetic biology, it is possible to improve existing organisms to process waste that is traditionally not used for biofuel production, such as whey.With the redesign of metabolic pathways, it is possible to create connections for the implementation of new organisms that carry out functions that are normally not present in nature.From a computational point of view, metabolic pathways, which can be found in data sources as KEGG, can be converted to a graph data structure. These transformations enable the use of well-known algorithms, which enables the optimization of the analyses required to achieve the assembly of new organisms.The present work aims to design a tool for the transformation of metabolic pathways and the development of path finding algorithms that establish relevant links between compounds that are essential to the biofuel production process.As a result, a catalog of biobricks is created from the analysis of a subset of paths which can be used in the design stage of the synthetic assembly of the E. coli bacteria. The assembly’s structure and functions are characterized according to the pieces used.Finally, new constructions are visualized with the goal of demonstrating and supporting the analysis processes, thus assisting people that work in the field of Synthetic Biology.AvailabilityPathways Analyzer is accessible at: https://gitlab.com/lvasquezcr/pathways-analyzer/

scholarly journals Novel two-stage processes for optimal chemical production in microbes

2019 ◽  
Author(s):  
Kaushik Raj ◽  
Naveen Venayak ◽  
Radhakrishnan Mahadevan
Keyword(s):  
Microbial Metabolism ◽  
Substrate Uptake ◽  
Chemical Production ◽  
Two Stage ◽  
Diverse Range ◽  
E Coli ◽  
Intermediate Growth ◽  
Wide Range ◽  
Production Stages ◽  
The Impact

AbstractMicrobial metabolism can be harnessed to produce a broad range of industrially important chemicals. Often, three key process variables: Titer, Rate and Yield (TRY) are the target of metabolic engineering efforts to improve microbial hosts toward industrial production. Previous research into improving the TRY metrics have examined the efficacy of having distinct growth and production stages to achieve enhanced productivity. However, these studies assumed a switch from a maximum growth to a maximum production phenotype. Hence, phenotypes with intermediate growth and chemical production for the growth and production stages of two-stage processes are yet to be explored. The impact of reduced growth rates on substrate uptake adds to the need for intelligent choice of operating points while designing two-stage processes. In this work, we develop a computational framework that scans the phenotypic space of microbial metabolism to identify ideal growth and production phenotypic targets, to achieve optimal TRY targets. Using this framework, with Escherichia coli as a model organism, we compare two-stage processes that use dynamic pathway regulation, with one-stage processes that use static intervention strategies, for different bioprocess objectives. Our results indicate that two-stage processes with intermediate growth during the production stage always result in optimal TRY values even in cases where substrate uptake is limited due to reduced growth during chemical production. By analyzing the flux distributions for the production enhancing strategies, we identify key reactions and reaction subsystems that require perturbation to achieve a production phenotype for a wide range of metabolites in E. coli. Interestingly, flux perturbations that increase phosphoenolpyruvate and NADPH availability are enriched among these production phenotypes. Furthermore, reactions in the pentose phosphate pathway emerge as key control nodes that function together to increase the availability of precursors to most products in E. coli. The inherently modular nature of microbial metabolism results in common reactions and reaction subsystems that need to be regulated to modify microbes from their target of growth to the production of a diverse range of metabolites. Due to the presence of these common patterns in the flux perturbations, we propose the possibility of a universal production strain.

Impact of Raw Materials and Processing Techniques on The Microbiological Quality of Egyptian Domiati Cheese

2020 ◽  
Keyword(s):  
Raw Materials ◽  
Microbiological Quality ◽  
Raw Milk ◽  
Plate Count ◽  
E Coli ◽  
Fresh Cheese ◽  
Processing Techniques ◽  
The Impact ◽  
Total Yeast

Domiati cheese is the most popular brand of cheese ripened in brine in the Middle East in terms of consumed quantities. This study was performed to investigate the impact of the microbiological quality of the used raw materials, the applied traditional processing techniques and ripening period on the quality and safety of the produced cheese. Three hundred random composite samples were collected from three factories at Fayoum Governorate, Egypt. Collected samples represent twenty-five each of: raw milk, table salt, calf rennet, microbial rennet, water, environmental air, whey, fresh cheese, ripened cheese & swabs from: worker hands; cheese molds and utensils; tanks. All samples were examined microbiologically for Standard Plate Count (SPC), coliforms count, Staphylococcus aureus (S. aureus) count, total yeast & mould count, presence of E. coli, Salmonellae and Listeria monocytogenes (L. monocytogenes). The mean value of SPC, coliforms, S. aureus and total yeast & mould counts ranged from (79×102 CFU/m3 for air to 13×108 CFU/g for fresh cheese), (7×102 MPN/ cm2 for tank swabs to 80×106 MPN/ml for raw milk), (9×102 CFU/g for salt to 69×106 CFU/g for fresh cheese) and (2×102 CFU/cm2 for hand swabs to 60×104 CFU/g for fresh cheese), respectively. Whereas, E. coli, Salmonella and L. monocytogenes failed to be detected in all examined samples. There were significant differences in all determined microbiological parameters (p ≤0.05) between fresh and ripened cheese which may be attributed to different adverse conditions such as water activity, pH, salt content and temperature carried out to improve the quality of the product.

scholarly journals KUALITAS AIR SUNGAI DI KAWASAN WISATA PANTAI PETITENGET, KEROBOKAN KABUPATEN BADUNG BALI

2018 ◽  
Vol 12 (2) ◽  
pp. 226
Author(s):  
Wayan Budiarsa Suyasa ◽  
Sri Kunti Pancadewi G. A ◽  
Iryanti E. Suprihatin ◽  
Dwi Adi Suastuti G. A.
Keyword(s):  
Water Quality ◽  
Structural Changes ◽  
Pollution Index ◽  
Basic Structure ◽  
Physical Damage ◽  
E Coli ◽  
Pollutant Source ◽  
Quality Category ◽  
Index Value ◽  
The Impact

In order to maintain the environmental carrying capacity of coastal tourism, this research was conducted to determine the condition of river water environmental pollution in the Petitenget beach area and pollutant source activities. Determination of water quality is carried out by analyzing the water quality taken at several sampling points in the four rivers that lead to the Petitenget beach. Determined the pollution index value (IP) of the physical chemical and biological pollution parameters. The results showed that the four rivers that flow into the Petitenget Beach area had been contaminated with indications of pH, BOD, COD, ammonia, Coliform and E. coli which exceeded water quality category III class quality (PerGub Bali No 16 Year 2016). The four rivers are included in the criteria of severe contamination. The four rivers have experienced physical damage or structural changes that have very high discharge fluctuations both in quantity and quality. Slimy basic structure, smelly and slum aesthetic waters. While the indication of the impact of pollution is waste water which is directly discharged into the river from hotels, restaurants, homestays, commercial centers and settlements.

scholarly journals Robust Inferences from a Before-and-After Study with Multiple Unaffected Control Groups

2013 ◽  
Vol 1 (2) ◽  
pp. 209-234 ◽  
Author(s):  
Pengyuan Wang ◽  
Mikhail Traskin ◽  
Dylan S. Small
Keyword(s):  
Sensitivity Analysis ◽  
Control Groups ◽  
Two Stage ◽  
Time Effects ◽  
Unaffected Control ◽  
Group Time ◽  
Before And After ◽  
Group Variation ◽  
The Impact ◽  
Before And After Study

AbstractThe before-and-after study with multiple unaffected control groups is widely applied to study treatment effects. The current methods usually assume that the control groups’ differences between the before and after periods, i.e. the group time effects, follow a normal distribution. However, there is usually no strong a priori evidence for the normality assumption, and there are not enough control groups to check the assumption. We propose to use a flexible skew-t distribution family to model group time effects, and consider a range of plausible skew-t distributions. Based on the skew-t distribution assumption, we propose a robust-t method to guarantee nominal significance level under a wide range of skew-t distributions, and hence make the inference robust to misspecification of the distribution of group time effects. We also propose a two-stage approach, which has lower power compared to the robust-t method, but provides an opportunity to conduct sensitivity analysis. Hence, the overall method of analysis is to use the robust-t method to test for the overall hypothesized range of shapes of group variation; if the test fails to reject, use the two-stage method to conduct a sensitivity analysis to see if there is a subset of group variation parameters for which we can be confident that there is a treatment effect. We apply the proposed methods to two datasets. One dataset is from the Current Population Survey (CPS) to study the impact of the Mariel Boatlift on Miami unemployment rates between 1979 and 1982.The other dataset contains the student enrollment and grade repeating data in West Germany in the 1960s with which we study the impact of the short school year in 1966–1967 on grade repeating rates.

scholarly journals The Location of Substitutions and Bacterial Genome Arrangements

2020 ◽  
Author(s):  
Daniella F Lato ◽  
G Brian Golding
Keyword(s):  
Sinorhizobium Meliloti ◽  
Bacterial Genome ◽  
Evolutionary Analysis ◽  
Origin Of Replication ◽  
Ancestral Reconstruction ◽  
Molecular Change ◽  
E Coli ◽  
A Genome ◽  
The Impact ◽  
Molecular Evolutionary Analysis

Abstract Increasing evidence supports the notion that different regions of a genome have unique rates of molecular change. This variation is particularly evident in bacterial genomes where previous studies have reported gene expression and essentiality tend to decrease, while substitution rates usually increases with increasing distance from the origin of replication. Genomic reorganization such as rearrangements occur frequently in bacteria and allow for the introduction and restructuring of genetic content, creating gradients of molecular traits along genomes. Here, we explore the interplay of these phenomena by mapping substitutions to the genomes of Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti, quantifying how many substitutions have occurred at each position in the genome. Preceding work indicates that substitution rate significantly increases with distance from the origin. Using a larger sample size and accounting for genome rearrangements through ancestral reconstruction, our analysis demonstrates that the correlation between the number of substitutions and distance from the origin of replication is often significant but small and inconsistent in direction. Some replicons had a significantly decreasing trend (E. coli and the chromosome of S. meliloti), while others showed the opposite significant trend (B. subtilis, Streptomyces, pSymA and pSymB in S. meliloti). dN, dS and ω were examined across all genes and there was no significant correlation between those values and distance from the origin. This study highlights the impact that genomic rearrangements and location have on molecular trends in some bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis. Assuming that molecular trends are exclusively in one direction can be problematic.

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