scholarly journals High-efficiency multi-site genomic editing (HEMSE) of Pseudomonas putida through thermoinducible ssDNA recombineering

2019 ◽  
Author(s):  
Tomas Aparicio ◽  
Akos Nyerges ◽  
Esteban Martínez-García ◽  
Víctor de Lorenzo
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Broad Host Range ◽  
Single Stranded Dna ◽  
Genetic Construct ◽  
Genomic Changes ◽  
Broad Host Range Plasmid ◽  
Set Up ◽  
Genomic Editing ◽  
Powerful Strategy

SUMMARYWhile single-stranded DNA recombineering is a powerful strategy for higher-scale genome editing, its application to species other than enterobacteria is typically limited by the efficiency of the recombinase and the action of native mismatch repair (MMR) systems. By building on [i] availability of the Erf-like single-stranded DNA-annealing protein Rec2, [ii] adoption of tightly-regulated thermoinducible device and [iii] transient expression of a MMR-supressing mutL allele, we have set up a coherent genetic platform for entering multiple changes in the chromosome of Pseudomononas putida with an unprecedented efficacy and reliability. The key genetic construct to this end is a broad host range plasmid encoding co-transcription of rec2 and P. putida’s mutLE36KPP at high levels under the control of the PL/cI857 system. Cycles of short thermal shifts of P. putida cells followed by transformation with a suite of mutagenic oligos delivered different types of high-fidelity genomic changes at frequencies up to 10% per single change—that can be handled without selection. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments—as shown in this case with ribosomal binding sites. These results enable the multiplexing of genome engineering of P. putida, as required for metabolic engineering of this important biotechnological chassis.

scholarly journals High-efficiency retron-mediated single-stranded DNA production in plants

2021 ◽  
Author(s):  
Wenjun Jiang ◽  
Gundra Sivakrishna Rao ◽  
Rashid Aman ◽  
Haroon Butt ◽  
Radwa Kamel ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genome Engineering ◽  
High Efficiency ◽  
Plant Biotechnology ◽  
Fold Increase ◽  
Gene Encoding ◽  
Single Stranded Dna ◽  
Homology Directed Repair ◽  
Potential Applications

ABSTRACT Background: Retrons are a class of retroelements that produce multicopy single-stranded DNA (msDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the over-production of single-stranded DNA (ssDNA), genome engineering, and directed evolution in bacteria, yeast, and mammalian cells. However, no studies have shown retron-mediated ssDNA production in plants, which could unlock potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Main methods and major results: Here, we demonstrated retron-mediated over-production of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated CRISPR-retron-coupled ssDNA over-production and targeted HDR in N. benthamiana. Conclusion: We present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.

scholarly journals The Conjugal Intermediate of Plasmid RSF1010 Inhibits Agrobacterium tumefaciens Virulence and VirB-Dependent Export of VirE2

1998 ◽  
Vol 180 (15) ◽  
pp. 3933-3939 ◽  
Author(s):  
Lisa E. Stahl ◽  
Amy Jacobs ◽  
Andrew N. Binns
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cell ◽  
Dna Binding Protein ◽  
Tumor Formation ◽  
Broad Host Range ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Crown Gall Disease ◽  
Broad Host Range Plasmid ◽  
Type Strains

ABSTRACT Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 betweenAgrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.

scholarly journals Chemical and Biological Molecules Involved in Differentiation, Maturation, and Survival of Dopaminergic Neurons in Health and Parkinson’s Disease: Physiological Aspects and Clinical Implications

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 754
Author(s):  
Giulia Gaggi ◽  
Andrea Di Credico ◽  
Pascal Izzicupo ◽  
Giovanni Iannetti ◽  
Angela Di Baldassarre ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Dopaminergic Neurons ◽  
Potential Role ◽  
High Efficiency ◽  
Biological Molecules ◽  
Alternative Approach ◽  
Non Coding Rnas ◽  
Set Up

Parkinson’s disease (PD) is one of the most common neurodegenerative disease characterized by a specific and progressive loss of dopaminergic (DA) neurons and dopamine, causing motor dysfunctions and impaired movements. Unfortunately, available therapies can partially treat the motor symptoms, but they have no effect on non-motor features. In addition, the therapeutic effect reduces gradually, and the prolonged use of drugs leads to a significative increase in the number of adverse events. For these reasons, an alternative approach that allows the replacement or the improved survival of DA neurons is very appealing for the treatment of PD patients and recently the first human clinical trials for DA neurons replacement have been set up. Here, we review the role of chemical and biological molecules that are involved in the development, survival and differentiation of DA neurons. In particular, we review the chemical small molecules used to differentiate different type of stem cells into DA neurons with high efficiency; the role of microRNAs and long non-coding RNAs both in DA neurons development/survival as far as in the pathogenesis of PD; and, finally, we dissect the potential role of exosomes carrying biological molecules as treatment of PD.

scholarly journals Sustainable Ventilation Strategies for a Medium-Sized Space with Regional Effect

2021 ◽  
Vol 13 (9) ◽  
pp. 4651
Author(s):  
Ming-Lun Alan Fong
Keyword(s):  
Life Cycle ◽  
Life Cycle Cost ◽  
Co2 Emission ◽  
High Efficiency ◽  
Regional Climate ◽  
Cost Effective ◽  
Regional Effect ◽  
Climate Conditions ◽  
Ventilation Strategies ◽  
Set Up

The analysis of ventilation strategies is fundamentally affected by regional climate conditions and local cost databases, in terms of energy consumption, CO2 emission and cost-effective analysis. A systematic approach is covered in this paper to estimate a local economic and environmental impact on a medium-sized space located in two regions during supply-and-installation and operation phases. Three ventilation strategies, including mixing ventilation (MV), displacement ventilation (DV) and stratum ventilation (SV) were applied to medium-sized air-conditioned space with this approach. The trend of the results for three ventilation systems in the life cycle assessment (LCA) and life cycle cost (LCC) analysis is SV < DV < MV. The result of CO2 emission and regional LCC shows that SV is the lowest one in both regional studies. In comparison with the Hong Kong Special Administrative Region (HKSAR) during 20 Service years, the case analysis demonstrates that the percentage differences in LCC analysis of MV, DV & SV in Guangdong are less than 20.5%, 19.4% and 18.82% respectively. Their CO2 emission of MV, DV and SV in Guangdong are more than HKSAR in 10.69%, 11.22% and 12.05%, respectively. The present study could provide information about regional effects in the LCA and LCC analysis of three ventilation strategies emissions, and thereby help set up models for decision-making on high efficiency and cost-effective ventilation strategy plans.

LPC Blade and Non-Axisymmetric Hub Profiling Optimization Using Multi-Fidelity Non-Intrusive POD Surrogates

2017 ◽  
Author(s):  
Tariq Benamara ◽  
Piotr Breitkopf ◽  
Ingrid Lepot ◽  
Caroline Sainvitu
Keyword(s):  
High Performance ◽  
High Efficiency ◽  
High Fidelity ◽  
Present Contribution ◽  
Compressor Blades ◽  
High Fidelity Simulations ◽  
Optimization Methodology ◽  
Set Up ◽  
Automated Optimization ◽  
Proper Orthogonal

The present contribution proposes a Reduced Order Model based multi-fidelity optimization methodology for the design of highly loaded blades in low pressure compressors. Environmental, as well as, economical limitations applied to engine manufacturers make the design of modern turbofans an extremely complex task. A smart compromise has to be found to guarantee both a high efficiency and a high average stage loading imposed for mass reduction constraints, while satisfying stability requirements. The design of compressor blades, usually involves at the same time a dedicated parametrization set-up in highdimensional space and high-fidelity simulations capturing, at least, efficiency and stability as most impacting phenomena. Despite recent advances in the high-performance computing area, introducing high-fidelity simulations into automated optimization, or even surrogate assisted optimization, loops still stands as a endeavor for engineers. In this framework, the proposed methodology is based on multi-fidelity surrogate models capable of representing the physics at hand in reduced spaces inferred from both precise, albeit costly, high-fidelity simulations and abundant, yet less accurate lower-fidelity data. Finally, we investigate the coupling of the proposed hierarchised multi-fidelity non-intrusive Proper Orthogonal Decomposition based surrogates with an evolutionary algorithm to reduce the number of high-fidelity simulation calls towards the targeted optimum.

Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control

1985 ◽  
pp. 173-188 ◽  
Author(s):  
Richard J. Meyer ◽  
Lung-Shen Lin ◽  
Kyunghoon Kim ◽  
Michael A. Brasch
Keyword(s):  
Host Range ◽  
Copy Number ◽  
Broad Host Range ◽  
Copy Number Control ◽  
Broad Host Range Plasmid
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