scholarly journals Construction and Enhancement of a Minimal Genetic AND Logic Gate

2008 ◽  
Vol 75 (3) ◽  
pp. 637-642 ◽  
Author(s):  
Daniel J. Sayut ◽  
Yan Niu ◽  
Lianhong Sun
Keyword(s):  
Transcriptional Activation ◽  
Logic Gate ◽  
Cellular Responses ◽  
Genetic Networks ◽  
Intercellular Signaling ◽  
Cellular Phenotype ◽  
Genetic Circuits ◽  
Regulatory Circuit ◽  
Shed Light ◽  
Activate Gene

ABSTRACT The ability of genetic networks to integrate multiple inputs in the generation of cellular responses is critical for the adaptation of cellular phenotype to distinct environments and of great interest in the construction of complex artificial circuits. To develop artificial genetic circuits that can integrate intercellular signaling molecules and commonly used inducing agents, we have constructed an artificial genetic AND gate based on the P luxI quorum-sensing promoter and the lac repressor. The hybrid promoter exhibited reduced basal and induced expression levels but increased expression capacity, generating clear logical responses that could be described using a simple mathematical model. The model also predicted that the AND gate's logic could be improved by altering the properties of the LuxR transcriptional activator and, in particular, by increasing its rate of transcriptional activation. Following these predictions, we were able to improve the AND gate's logic by ∼1.5-fold using a LuxR mutant library generated by directed evolution, providing the first example of the use of mutant transcriptional activators to improve the logic of a complex regulatory circuit. In addition, detailed characterizations of the AND gate's responses shed light on how LuxR, LacI, and RNA polymerase interact to activate gene expression.

scholarly journals Inhibition of Early Response Genes Prevents Changes in Global Joint Metabolomic Profiles in Mouse Post-Traumatic Osteoarthritis

2018 ◽  
Author(s):  
Dominik R. Haudenschild ◽  
Alyssa K. Carlson ◽  
Donald L. Zignego ◽  
Jasper H.N. Yik ◽  
Jonathan K. Hilmer ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Cellular Responses ◽  
Early Response ◽  
Primary Response ◽  
Response Gene ◽  
Joint Injury ◽  
Non Invasive ◽  
Primary Response Gene ◽  
Post Traumatic ◽  
Early Response Genes

Osteoarthritis (OA) is the most common degenerative joint disease, and joint injury increases the risk of OA by 10-fold. Although the injury event itself damages joint tissues, a substantial amount of secondary damage is mediated by the cellular responses to the injury. Cellular responses include the production and activation of proteases (MMPs, ADAMTSs, Cathepsins), the production of inflammatory cytokines, and we hypothesize, changes to the joint metabolome. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Flavopiridol is a potent and selective inhibitor of Cdk9 kinase activity, which prevents the transcriptional activation of early response genes. To model post-traumatic osteoarthritis, we subjected mice to non-invasive ACL-rupture joint injury. Following injury, mice were treated with flavopiridol to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1 hour after injury. We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism and others. Importantly, we found that inhibition of primary response gene activation at the time of injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites, which may offer novel targets for cell-mediated secondary joint damage. Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses, and these data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury.Significance StatementJoint injury is an excellent predictor of future osteoarthritis. It is increasingly apparent that the acute cellular responses to injury contribute to the initiation and pathogenesis of OA. Although changes to the joint transcriptome have been extensively studied in the context of joint injury, little is known about changes to small-molecule metabolites. Here we use a non-invasive ACL rupture model of joint injury in mice to identify injury-induced changes to the global metabolomic profiles. In one experimental group we prevented the activation of primary response gene transcription using the Cdk9 inhibitor flavopiridol. Through this comparison, we identified two sets of metabolites that change acutely after joint injury: those that require transcription of primary response genes, and those that do not.

scholarly journals A conserved regulatory module regulates receptor kinase signaling in immunity and development

2021 ◽  
Author(s):  
Thomas A. DeFalco ◽  
Pauline Anne ◽  
Sean R. James ◽  
Andrew Willoughby ◽  
Oliver Johanndrees ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cellular Responses ◽  
Receptor Signaling ◽  
Receptor Kinase ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Regulatory Circuit ◽  
Regulatory Module ◽  
Molecular Patterns ◽  
Receptor Kinase Signaling

ABSTRACTLigand recognition by cell-surface receptors underlies development and immunity in both animals and plants. Modulating receptor signaling is critical for appropriate cellular responses but the mechanisms ensuring this are poorly understood. Here, we show that signaling by plant receptors for pathogen-associated molecular patterns (PAMPs) in immunity and CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptides (CLEp) in development employ a similar regulatory module. In the absence of ligand, signaling is dampened through association with specific type-2C protein phosphatases (PP2Cs). Upon activation, PAMP and CLEp receptors phosphorylate divergent cytosolic kinases, which, in turn, phosphorylate the phosphatases, thereby promoting their release from the receptor complexes. Our work reveals a regulatory circuit shared between immune and developmental receptor signaling, which may have broader important implications for plant receptor kinase-mediated signaling in general.

scholarly journals Multiplexed Cell-Based Diagnostic Devices for Detection of Renal Biomarkers Using Genetic Circuits

2021 ◽  
Author(s):  
Sila Kose ◽  
Recep Ahan ◽  
Ilkay Koksaldi ◽  
Muazzez Olgac ◽  
Cigdem Kasapkara ◽  
...  
Keyword(s):  
Control Strategies ◽  
Low Cost ◽  
Logic Gate ◽  
Genetic Circuits ◽  
Major Health ◽  
Expression Control ◽  
Renal Biomarkers ◽  
Gene Expression Control ◽  
Response Profiles ◽  
Operational Range

The number of synthetic biology based solutions employed in the medical industry is growing every year. The whole cell biosensors being one of them, have been proven valuable tools for developing low-cost, portable, personalized medicine alternatives to conventional techniques. Based on this concept, we targeted one of the major health problems in the world, Chronic Kidney Disease (CKD). To do so, we developed two novel biosensors for the detection of two important renal biomarkers; urea and uric acid. Using advanced gene expression control strategies we improved the operational range and the response profiles of each biosensor to meet clinical specifications. We further engineered these systems to enable multiplexed detection as well as an AND-logic gate operating system. Finally, we tested the applicability of these systems and optimized their working dynamics inside complex medium human blood serum. This study could help the efforts to transition from labor-intensive and expensive laboratory techniques to widely available, portable, low cost diagnostic options.

scholarly journals Computational modelling of cambium activity provides a regulatory framework for simulating radial plant growth

2020 ◽  
Author(s):  
Ivan Lebovka ◽  
Bruno Hay Mele ◽  
Alexandra Zakieva ◽  
Nial Gursanscky ◽  
Roeland Merks ◽  
...  
Keyword(s):  
Fundamental Problem ◽  
Computational Modelling ◽  
Intercellular Signaling ◽  
Tissue Organization ◽  
Regulatory Circuit ◽  
Number Of Factors ◽  
A Cell ◽  
Cambium Activity ◽  
Cell Niche ◽  
Terrestrial Biomass

AbstractPrecise organization of growing structures is a fundamental problem in developmental biology. In plants, radial growth is mediated by the cambium, a stem cell niche continuously producing wood (xylem) and bast (phloem) in a strictly bidirectional manner. While this process contributes large parts to terrestrial biomass, cambium dynamics eludes direct experimental access due to obstacles in live cell imaging. Here, we present a cell-based computational model visualizing cambium activity and integrating the function of central cambium regulators. Performing iterative comparisons of plant and model anatomies, we conclude that an intercellular signaling module consisting of the receptor-like kinase PXY and its ligand CLE41 constitutes a minimal framework sufficient for instructing tissue organization. Employing genetically encoded markers for different cambium domains in backgrounds with altered PXY/CLE41 activity, we furthermore propose that the module is part of a larger regulatory circuit using the phloem as a morphogenetic center. Our model highlights the importance of intercellular communication along the radial sequence of tissues within the cambium area and shows that a limited number of factors is sufficient to create a stable bidirectional tissue production.

Lysine methylation and the regulation of p53

2012 ◽  
Vol 52 ◽  
pp. 79-92 ◽  
Author(s):  
Simon M. Carr ◽  
Shonagh Munro ◽  
Nicholas B. La Thangue
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Genotoxic Stress ◽  
Cellular Responses ◽  
Future Research ◽  
Lysine Methylation ◽  
Post Translational Modifications ◽  
Cycle Arrest ◽  
Tumour Suppressor Protein ◽  
Protein Functions

The p53 tumour suppressor protein functions as a guardian against genotoxic stress. This function is mediated in part by the transcriptional activation of genes involved in cell-cycle arrest, apoptosis, DNA repair and autophagy. The activity of p53 is regulated by a complex array of post-translational modifications, which function as a code to determine cellular responses to a given stress. In this chapter we highlight recent advances in our understanding of this code, with particular reference to lysine methylation, and discuss implications for future research.

scholarly journals Gat1p, a GATA family protein whose production is sensitive to nitrogen catabolite repression, participates in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (3) ◽  
pp. 847-858 ◽  
Author(s):  
J A Coffman ◽  
R Rai ◽  
T Cunningham ◽  
V Svetlov ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Catabolite Repression ◽  
Nitrogen Sources ◽  
Nitrogen Catabolite Repression ◽  
Triple Mutant ◽  
Regulatory Circuit ◽  
Catabolic Genes ◽  
Family Protein ◽  
Sequence Elements

Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.

scholarly journals Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase.

1988 ◽  
Vol 8 (5) ◽  
pp. 2132-2139 ◽  
Author(s):  
I Roussou ◽  
G Thireos ◽  
B M Hauge
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Protein Kinase ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Translational Regulation ◽  
Catalytic Domain ◽  
Amino Acid Starvation ◽  
Protein Kinase Activity ◽  
Regulatory Circuit

GCN4 protein mediates the transcriptional activation of amino acid biosynthetic genes in Saccharomyces cerevisiae by specifically binding to DNA sequences in their 5'-regulatory regions. GCN4 expression is regulated at the level of translation, with translational derepression occurring under conditions of amino acid starvation. The product of the GCN2 gene is essential for translational derepression of GCN4. Sequence analysis of the GCN2 gene reveals that the GCN2 protein has a domain highly homologous to the catalytic domain of all known protein kinases. Furthermore, gcn2 strains are deficient in a protein kinase activity corresponding to a protein with the calculated molecular weight deduced from the GCN2 open reading frame. Therefore it is likely that GCN2 encodes a protein kinase, which may be directly involved in translational regulation of the GCN4 mRNA. Transcription of the GCN2 gene is increased when cells are cultured in amino acid starvation medium. This transcriptional activation is mediated by the GCN4 protein, which binds to the promoter region of the GCN2 gene. Thus, this system is modulated by a transcriptional-translational regulatory circuit, which is activated by amino acid starvation. Activation is not the result of a simple quantitative increase of either one of the identified components of the circuit.

scholarly journals Interactions of nanoparticles with pulmonary structures and cellular responses

2008 ◽  
Vol 294 (5) ◽  
pp. L817-L829 ◽  
Author(s):  
Christian Mühlfeld ◽  
Barbara Rothen-Rutishauser ◽  
Fabian Blank ◽  
Dimitri Vanhecke ◽  
Matthias Ochs ◽  
...  
Keyword(s):  
Inhalation Exposure ◽  
Experimental Studies ◽  
Cellular Responses ◽  
Epidemiological Studies ◽  
Oxygen Species ◽  
Adverse Health Effects ◽  
Cardiovascular Morbidity And Mortality ◽  
Dose Metrics ◽  
Methodological Problems ◽  
Shed Light

Combustion-derived and synthetic nano-sized particles (NSP) have gained considerable interest among pulmonary researchers and clinicians for two main reasons. 1) Inhalation exposure to combustion-derived NSP was associated with increased pulmonary and cardiovascular morbidity and mortality as suggested by epidemiological studies. Experimental evidence has provided a mechanistic picture of the adverse health effects associated with inhalation of combustion-derived and synthetic NSP. 2) The toxicological potential of NSP contrasts with the potential application of synthetic NSP in technological as well as medicinal settings, with the latter including the use of NSP as diagnostics or therapeutics. To shed light on this paradox, this article aims to highlight recent findings about the interaction of inhaled NSP with the structures of the respiratory tract including surfactant, alveolar macrophages, and epithelial cells. Cellular responses to NSP exposure include the generation of reactive oxygen species and the induction of an inflammatory response. Furthermore, this review places special emphasis on methodological differences between experimental studies and the caveats associated with the dose metrics and points out ways to overcome inherent methodological problems.

Dual phosphorylation in response regulator protein PrrA is crucial for intracellular survival of mycobacteria consequent upon transcriptional activation

2017 ◽  
Vol 474 (24) ◽  
pp. 4119-4136 ◽  
Author(s):  
Alok K. Mishra ◽  
Shivraj M. Yabaji ◽  
Rikesh K. Dubey ◽  
Ekta Dhamija ◽  
Kishore K. Srivastava
Keyword(s):  
Transcriptional Activation ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Peritoneal Macrophages ◽  
Physical Interaction ◽  
Fast Growing ◽  
Regulatory Circuit ◽  
Regulator Protein ◽  
Promoter Dna

The remarkable ability of Mycobacterium tuberculosis (Mtb) to survive inside human macrophages is attributed to the presence of a complex sensory and regulatory network. PrrA is a DNA-binding regulatory protein, belonging to an essential two-component system (TCS), PrrA/B, which is required for early phase intracellular replication of Mtb. Despite its importance, the mechanism of PrrA/B-mediated signaling is not well understood. In the present study, we demonstrate that the binding of PrrA on the promoter DNA and its consequent activation is cumulatively controlled via dual phosphorylation of the protein. We have further characterized the role of terminal phospho-acceptor domain in the physical interaction of PrrA with its cognate kinase PrrB. The genetic deletion of prrA/B in Mycobacterium smegmatis was possible only in the presence of ectopic copies of the genes, suggesting the essentiality of this TCS in fast-growing mycobacterial strains as well. The overexpression of phospho-mimetic mutant (T6D) altered the growth of M. smegmatis in an in vitro culture and affected the replication of Mycobacterium bovis BCG in mouse peritoneal macrophages. Interestingly, the Thr6 site was found to be conserved in Mtb complex, whereas it was altered in some fast-growing mycobacterial strains, indicating that this unique phosphorylation might be predominant in employing the regulatory circuit in M. bovis BCG and presumably also in Mtb complex.

scholarly journals Enhanced regulation of prokaryotic gene expression by a eukaryotic transcriptional activator

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
I. Cody MacDonald ◽  
Travis R. Seamons ◽  
Jonathan C. Emmons ◽  
Shwan B. Javdan ◽  
Tara L. Deans
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Dynamic Range ◽  
Current Trend ◽  
Medical Science ◽  
Genetic Circuits ◽  
Eukaryotic Transcription ◽  
Complex Functions ◽  
Eukaryotic Gene

AbstractExpanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine. Here, we reverse the current trend of moving genetic parts from prokaryotes to eukaryotes and demonstrate that the activating eukaryotic transcription factor QF and its corresponding DNA-binding sequence can be moved to E. coli to introduce transcriptional activation, in addition to tight off states. We further demonstrate that the QF transcription factor can be used in genetic devices that respond to low input levels with robust and sustained output signals. Collectively, we show that eukaryotic gene regulator elements are functional in prokaryotes and establish a versatile and broadly applicable approach for constructing genetic circuits with complex functions. These genetic tools hold the potential to improve biotechnology applications for medical science and research.

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