scholarly journals Efficient Search, Mapping, and Optimization of Multi-protein Genetic Systems in Diverse Bacteria

2013 ◽  
Author(s):  
Iman Farasat ◽  
Manish Kushwaha ◽  
Jason Collens ◽  
Michael Easterbrook ◽  
Matthew Guido ◽  
...  
Keyword(s):  
Predictive Models ◽  
Search Algorithm ◽  
Genetic System ◽  
System Level ◽  
Response Curves ◽  
Combinatorial Explosion ◽  
Genetic Systems ◽  
Expression Activity ◽  
Rate Limiting

Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a >10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs.

scholarly journals Stability and Robustness of Unbalanced Genetic Toggle Switches in the Presence of Scarce Resources

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 271
Author(s):  
Chentao Yong ◽  
Andras Gyorgy
Keyword(s):  
Cell Fate ◽  
Rational Design ◽  
Resource Competition ◽  
Dynamic Properties ◽  
Mechanistic Model ◽  
Robustness Analysis ◽  
System Level ◽  
Genetic Circuits ◽  
Scarce Resources ◽  
Genetic Systems

While the vision of synthetic biology is to create complex genetic systems in a rational fashion, system-level behaviors are often perplexing due to the context-dependent dynamics of modules. One major source of context-dependence emerges due to the limited availability of shared resources, coupling the behavior of disconnected components. Motivated by the ubiquitous role of toggle switches in genetic circuits ranging from controlling cell fate differentiation to optimizing cellular performance, here we reveal how their fundamental dynamic properties are affected by competition for scarce resources. Combining a mechanistic model with nullcline-based stability analysis and potential landscape-based robustness analysis, we uncover not only the detrimental impacts of resource competition, but also how the unbalancedness of the switch further exacerbates them. While in general both of these factors undermine the performance of the switch (by pushing the dynamics toward monostability and increased sensitivity to noise), we also demonstrate that some of the unwanted effects can be alleviated by strategically optimized resource competition. Our results provide explicit guidelines for the context-aware rational design of toggle switches to mitigate our reliance on lengthy and expensive trial-and-error processes, and can be seamlessly integrated into the computer-aided synthesis of complex genetic systems.

scholarly journals Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

2021 ◽  
Vol 22 (2) ◽  
pp. 734
Author(s):  
Paul K. Varghese ◽  
Mones Abu-Asab ◽  
Emilios K. Dimitriadis ◽  
Monika B. Dolinska ◽  
George P. Morcos ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Large Scale ◽  
Magnetic Beads ◽  
Enzymatic Reaction ◽  
Oculocutaneous Albinism ◽  
Transmission Electron ◽  
Hill Kinetics ◽  
Rate Limiting ◽  
Human Tyrosinase

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19–469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

At-wavelength, system-level flare characterization of extreme-ultraviolet optical systems

2000 ◽  
Vol 39 (17) ◽  
pp. 2941 ◽  
Author(s):  
Patrick Naulleau ◽  
Kenneth A. Goldberg ◽  
Eric M. Gullikson ◽  
Jeffrey Bokor
Keyword(s):  
Extreme Ultraviolet ◽  
System Level ◽  
Optical Systems

Characterization of Glucose Response Curves after Insulin Injection in Sensitive versus Insensitive Mares

2013 ◽  
Vol 33 (11) ◽  
pp. 937-941 ◽  
Author(s):  
Jeanne D. Lestelle ◽  
Donald L. Thompson ◽  
Rebekah C. Hebert
Keyword(s):  
Insulin Injection ◽  
Response Curves ◽  
Glucose Response

Application of Atom Probe Microanalysis for Understanding Microstructure Evolution in Nickel Base Superalloy Welds

2000 ◽  
Vol 6 (S2) ◽  
pp. 350-351
Author(s):  
S. S. Babu ◽  
S. A. David ◽  
M. K. Miller
Keyword(s):  
Microstructure Evolution ◽  
Predictive Models ◽  
Strong Relationship ◽  
Atom Probe ◽  
Systematic Investigation ◽  
Nickel Base Superalloy ◽  
Cooling Conditions ◽  
Nickel Base ◽  
Base Superalloy

The characterization of the microstructure evolution during welding of nickel base superalloys is required for efficient reuse and reclamation of used and failed components. Previous atom probe analysis of electron-beam and laser-beam welds revealed complex alloying elemental partitioning between the γ and γ phases. Rapid cooling conditions in the weld leads to non-equilibrium partitioning and large amplitude Cr and Co levels in the γ phase. These results indicated that there is a strong relationship between weld cooling rate and the precipitation of γ′ precipitates from the γ phase. To understand and develop predictive models, a systematic investigation of the microstructure evolution in CM247DS alloy under controlled thermomechanical conditions are being performed. This paper describes some recent results on the elemental partitioning between γ and γ′ phases obtained with atom probe microanalysis.

scholarly journals The Orphan Crop Crassocephalum crepidioides Accumulates the Pyrrolizidine Alkaloid Jacobine in Response to Nitrogen Starvation

2021 ◽  
Vol 12 ◽  
Author(s):  
Sebastian Schramm ◽  
Wilfried Rozhon ◽  
Adebimpe N. Adedeji-Badmus ◽  
Yuanyuan Liang ◽  
Shahran Nayem ◽  
...  
Keyword(s):  
Pyrrolizidine Alkaloid ◽  
Sub Saharan Africa ◽  
Close Relative ◽  
Environmental Cues ◽  
N Deficiency ◽  
Sub Saharan ◽  
Rate Limiting ◽  
Homospermidine Synthase ◽  
Orphan Crop

Crassocephalum crepidioides is an African orphan crop that is used as a leafy vegetable and medicinal plant. Although it is of high regional importance in Sub-Saharan Africa, the plant is still mainly collected from the wild and therefore efforts are made to promote its domestication. However, in addition to beneficial properties, there was first evidence that C. crepidioides can accumulate the highly toxic pyrrolizidine alkaloid (PA) jacobine and here it was investigated, how jacobine production is controlled. Using ecotypes from Africa and Asia that were characterized in terms of their PA profiles, it is shown that the tetraploid C. crepidioides forms jacobine, an ability that its diploid close relative Crassocephalum rubens appears to lack. Evidence is provided that nitrogen (N) deficiency strongly increases jacobine in the leaves of C. crepidioides, that this capacity depends more strongly on the shoot than the root system, and that homospermidine synthase (HSS) activity is not rate-limiting for this reaction. A characterization of HSS gene representation and transcription showed that C. crepidioides and C. rubens possess two functional versions, one of which is conserved, that the HSS transcript is mainly present in roots and that its abundance is not controlled by N deficiency. In summary, this work improves our understanding of how environmental cues impact PA biosynthesis in plants and provides a basis for the development of PA-free C. crepidioides cultivars, which will aid its domestication and safe use.

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