scholarly journals An automated model test system for systematic development and improvement of gene expression models

2017 ◽  
Author(s):  
Alexander C. Reis ◽  
Howard M. Salis
Keyword(s):  
Gene Expression ◽  
Model Test ◽  
Translation Initiation ◽  
Test System ◽  
Biophysical Model ◽  
Genetic Circuits ◽  
Initiation Rate ◽  
Information Theoretic ◽  
New Information ◽  
Genetic Systems

ABSTRACTGene expression models greatly accelerate the engineering of synthetic metabolic pathways and genetic circuits by predicting sequence-function relationships and reducing trial-and-error experimentation. However, developing models with more accurate predictions is a significant challenge, even though they are essential to engineering complex genetic systems. Here we present a model test system that combines advanced statistics, machine learning, and a database of 9862 characterized genetic systems to automatically quantify model accuracies, accept or reject mechanistic hypotheses, and identify areas for model improvement. We also introduce Model Capacity, a new information theoretic metric that enables correct model comparisons across datasets. We demonstrate the model test system by comparing six models of translation initiation rate, evaluating 100 mechanistic hypotheses, and uncovering new sequence determinants that control protein expression levels. We applied these results to develop a biophysical model of translation initiation rate with significant improvements in accuracy. Automated model test systems will dramatically accelerate the development of gene expression models, and thereby transition synthetic biology into a mature engineering discipline.

scholarly journals Fine-tuning biosensor dynamic range based on rational design of cross-ribosome-binding sites in bacteria

2020 ◽  
Author(s):  
Nana Ding ◽  
Shenghu Zhou ◽  
Zhenqi Yuan ◽  
Xiaojuan Zhang ◽  
Jing Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Deep Learning ◽  
Translation Initiation ◽  
Rational Design ◽  
Dynamic Range ◽  
Regulatory Elements ◽  
Fine Tuning ◽  
Initiation Rate ◽  
Ribosome Binding

ABSTRACTCurrently, predictive translation tuning of regulatory elements to the desired output of transcription factor based biosensors remains a challenge. The gene expression of a biosensor system must exhibit appropriate translation intensity, which is controlled by the ribosome-binding site (RBS), to achieve fine-tuning of its dynamic range (i.e., fold change in gene expression between the presence and absence of inducer) by adjusting the translation initiation rate of the transcription factor and reporter. However, existing genetically encoded biosensors generally suffer from unpredictable translation tuning of regulatory elements to dynamic range. Here, we elucidated the connections and partial mechanisms between RBS, translation initiation rate, protein folding and dynamic range, and presented a rational design platform that predictably tuned the dynamic range of biosensors based on deep learning of large datasets cross-RBSs (cRBSs). A library containing 24,000 semi-rationally designed cRBSs was constructed using DNA microarray, and was divided into five sub-libraries through fluorescence-activated cell sorting. To explore the relationship between cRBSs and dynamic range, we established a classification model with the cRBSs and average dynamic range of five sub-libraries to accurately predict the dynamic range of biosensors based on convolutional neural network in deep learning. Thus, this work provides a powerful platform to enable predictable translation tuning of RBS to the dynamic range of biosensors.

scholarly journals Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites

2013 ◽  
Vol 42 (4) ◽  
pp. 2646-2659 ◽  
Author(s):  
Amin Espah Borujeni ◽  
Anirudh S. Channarasappa ◽  
Howard M. Salis
Keyword(s):  
Free Energy ◽  
Transcriptional Regulation ◽  
Translation Initiation ◽  
Context Effects ◽  
Binding Free Energy ◽  
Biophysical Model ◽  
Initiation Rate ◽  
Translation Rate ◽  
Trade Offs ◽  
Post Transcriptional Regulation

Abstract The ribosome’s interactions with mRNA govern its translation rate and the effects of post-transcriptional regulation. Long, structured 5′ untranslated regions (5′ UTRs) are commonly found in bacterial mRNAs, though the physical mechanisms that determine how the ribosome binds these upstream regions remain poorly defined. Here, we systematically investigate the ribosome’s interactions with structured standby sites, upstream of Shine–Dalgarno sequences, and show that these interactions can modulate translation initiation rates by over 100-fold. We find that an mRNA’s translation initiation rate is controlled by the amount of single-stranded surface area, the partial unfolding of RNA structures to minimize the ribosome’s binding free energy penalty, the absence of cooperative binding and the potential for ribosomal sliding. We develop a biophysical model employing thermodynamic first principles and a four-parameter free energy model to accurately predict the ribosome’s translation initiation rates for 136 synthetic 5′ UTRs with large structures, diverse shapes and multiple standby site modules. The model predicts and experiments confirm that the ribosome can readily bind distant standby site modules that support high translation rates, providing a physical mechanism for observed context effects and long-range post-transcriptional regulation.

scholarly journals A suppressor tRNA-mediated feedforward loop eliminates leaky gene expression in bacteria

2020 ◽  
Author(s):  
Joanne M L Ho ◽  
Corwin A Miller ◽  
Sydney E Parks ◽  
Jacob R Mattia ◽  
Matthew R Bennett
Keyword(s):  
Gene Expression ◽  
Cancer Detection ◽  
Background Subtraction ◽  
Fold Change ◽  
Genetic Circuits ◽  
Persistent Problem ◽  
Suppressor Trna ◽  
Feedforward Loop ◽  
Genetic Systems ◽  
Fold Induction

Abstract Ligand-inducible genetic systems are the mainstay of synthetic biology, allowing gene expression to be controlled by the presence of a small molecule. However, ‘leaky’ gene expression in the absence of inducer remains a persistent problem. We developed a leak dampener tool that drastically reduces the leak of inducible genetic systems while retaining signal in Escherichia coli. Our system relies on a coherent feedforward loop featuring a suppressor tRNA that enables conditional readthrough of silent non-sense mutations in a regulated gene, and this approach can be applied to any ligand-inducible transcription factor. We demonstrate proof-of-principle of our system with the lactate biosensor LldR and the arabinose biosensor AraC, which displayed a 70-fold and 630-fold change in output after induction of a fluorescence reporter, respectively, without any background subtraction. Application of the tool to an arabinose-inducible mutagenesis plasmid led to a 540-fold change in its output after induction, with leak decreasing to the level of background mutagenesis. This study provides a modular tool for reducing leak and improving the fold-induction within genetic circuits, demonstrated here using two types of biosensors relevant to cancer detection and genetic engineering.

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.

Development and preliminary application of a plate loading model test system considering stress state

Measurement ◽  
2021 ◽  
pp. 109507
Author(s):  
Changqi Zhu ◽  
Xing Wang ◽  
Mingjian Hu ◽  
Xinzhi Wang ◽  
Jianhua Shen ◽  
...  
Keyword(s):  
Stress State ◽  
Model Test ◽  
Test System ◽  
Preliminary Application ◽  
Plate Loading

scholarly journals High-performance chemical- and light-inducible recombinases in mammalian cells and mice

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin H. Weinberg ◽  
Jang Hwan Cho ◽  
Yash Agarwal ◽  
N. T. Hang Pham ◽  
Leidy D. Caraballo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Performance ◽  
Genome Engineering ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Expression Control ◽  
Gene Expression Control ◽  
High Performance Systems ◽  
Spatiotemporal Control

Abstract Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

scholarly journals The Role of Translation Initiation Regulation in Haematopoiesis

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Godfrey Grech ◽  
Marieke von Lindern
Keyword(s):  
Gene Expression ◽  
Translation Initiation ◽  
Translational Control ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Mrna Translation ◽  
Translation Control ◽  
Haematological Disorders

Organisation of RNAs into functional subgroups that are translated in response to extrinsic and intrinsic factors underlines a relatively unexplored gene expression modulation that drives cell fate in the same manner as regulation of the transcriptome by transcription factors. Recent studies on the molecular mechanisms of inflammatory responses and haematological disorders indicate clearly that the regulation of mRNA translation at the level of translation initiation, mRNA stability, and protein isoform synthesis is implicated in the tight regulation of gene expression. This paper outlines how these posttranscriptional control mechanisms, including control at the level of translation initiation factors and the role of RNA binding proteins, affect hematopoiesis. The clinical relevance of these mechanisms in haematological disorders indicates clearly the potential therapeutic implications and the need of molecular tools that allow measurement at the level of translational control. Although the importance of miRNAs in translation control is well recognised and studied extensively, this paper will exclude detailed account of this level of control.

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