scholarly journals Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits

2019 ◽  
Author(s):  
Sally Wang ◽  
Gregory F. Payne ◽  
William E. Bentley
Keyword(s):  
Quorum Sensing ◽  
Genetic Circuits ◽  
E Coli

Design of Synthetic Quorum Sensing Achieving Induction Timing-Independent Signal Stabilization for Dynamic Metabolic Engineering of E. coli

2021 ◽  
Author(s):  
Yuki Soma ◽  
Masatomo Takahashi ◽  
Yuri Fujiwara ◽  
Tamaki Shinohara ◽  
Yoshihiro Izumi ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Quorum Sensing ◽  
E Coli

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals A MATLAB Toolbox for Modeling Genetic Circuits in Cell-Free Systems

2020 ◽  
Author(s):  
Vipul Singhal ◽  
Zoltan A. Tuza ◽  
Zachary Z. Sun ◽  
Richard M. Murray
Keyword(s):  
Reaction Rate ◽  
Behavior Prediction ◽  
Genetic Circuits ◽  
Matlab Toolbox ◽  
Parameter Inference ◽  
Batch Mode ◽  
E Coli ◽  
Entire Duration ◽  
Resource Loading

AbstractWe introduce a MATLAB based simulation toolbox, called txtlsim, for an E. coli based Transcription-Translation (TX-TL) system. This toolbox accounts for several cell-free related phenomena, such as resource loading, consumption, and degradation, and in doing so, models the dynamics of TX-TL reactions for the entire duration of batch-mode experiments. We use a Bayesian parameter inference approach to characterize the reaction rate parameters associated with the core transcription, translation and mRNA degradation mechanics of the toolbox, allowing it to reproduce constitutive mRNA and protien expression trajectories. We demonstrate the use of this characterized toolbox in a circuit behavior prediction case study for an incoherent feed-forward loop.

scholarly journals Programmable Microbial Ink for 3D Printing of Living Materials Produced from Genetically Engineered Protein Nanofibers

2021 ◽  
Author(s):  
Anna M Duraj-Thatte ◽  
Avinash Manjula Basavanna ◽  
Jarod Rutledge ◽  
Jing Xia ◽  
Shabir Hassan ◽  
...  
Keyword(s):  
3D Printing ◽  
Self Assembly ◽  
Genetically Engineered ◽  
Ambient Conditions ◽  
Chemical Induction ◽  
Genetic Circuits ◽  
3D Print ◽  
E Coli ◽  
Molecular Components ◽  
Living Materials

Living cells have the capability to synthesize molecular components and precisely assemble them from the nanoscale to build macroscopic living functional architectures under ambient conditions. The emerging field of living materials has leveraged microbial engineering to produce materials for various applications, but building 3D structures in arbitrary patterns and shapes has been a major challenge. We set out to develop a new bioink, termed as "microbial ink" that is produced entirely from genetically engineered microbial cells, programmed to perform a bottom-up, hierarchical self-assembly of protein monomers into nanofibers, and further into nanofiber networks that comprise extrudable hydrogels. We further demonstrate the 3D printing of functional living materials by embedding programmed Escherichia coli (E. coli) cells and nanofibers into microbial ink, which can sequester toxic moieties, release biologics and regulate its own cell growth through the chemical induction of rationally designed genetic circuits. This report showcases the advanced capabilities of nanobiotechnology and living materials technology to 3D-print functional living architectures.

scholarly journals Novel Quorum-Sensing Peptides Mediating Interspecies Bacterial Cell Death

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Sathish Kumar ◽  
Ilana Kolodkin-Gal ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Bacterial Cell ◽  
Content Type ◽  
E Coli ◽  
New Class ◽  
Bacterial Cell Death ◽  
Induced Module

ABSTRACTEscherichia colimazEFis a toxin-antitoxin stress-induced module mediating cell death. It requires the quorum-sensing signal (QS) “extracellular death factor” (EDF), the penta-peptide NNWNN (EcEDF), enhancing the endoribonucleolytic activity ofE. colitoxin MazF. Here we discovered thatE. coli mazEF-mediated cell death could be triggered by QS peptides from the supernatants (SN) of the Gram-positive bacteriumBacillus subtilisand the Gram-negative bacteriumPseudomonas aeruginosa. In the SN ofB. subtilis, we found one EDF, the hexapeptide RGQQNE, calledBsEDF. In the SN ofP. aeruginosa, we found three EDFs: the nonapeptide INEQTVVTK, calledPaEDF-1, and two hexadecapeptides, VEVSDDGSGGNTSLSQ, calledPaEDF-2, and APKLSDGAAAGYVTKA, calledPaEDF-3. When added to a dilutedE. colicultures, each of these peptides acted as an interspecies EDF that triggeredmazEF-mediated death. Furthermore, though their sequences are very different, each of these EDFs amplified the endoribonucleolytic activity ofE. coliMazF, probably by interacting with different sites onE. coliMazF. Finally, we suggest that EDFs may become the basis for a new class of antibiotics that trigger death from outside the bacterial cells.IMPORTANCEBacteria communicate with one another via quorum-sensing signal (QS) molecules. QS provides a mechanism for bacteria to monitor each other’s presence and to modulate gene expression in response to population density. Previously, we addedE. coliEDF (EcEDF), the peptide NNWNN, to this list of QS molecules. Here we extended the group of QS peptides to several additional different peptides. The new EDFs are produced by two other bacteria,Bacillus subtilisandPseudomonas aeruginosa. Thus, in this study we established a “new family of EDFs.” This family provides the first example of quorum-sensing molecules participating in interspecies bacterial cell death. Furthermore, each of these peptides provides the basis of a new class of antibiotics triggering death by acting from outside the cell.

scholarly journals Virtual Screening of SdiA Inhibitors from Melia dubia to Curtail Uropathogenic E. coli Quorum Sensing

2013 ◽  
Vol 25 (1) ◽  
pp. 95-100 ◽  
Author(s):  
Vinothkannan Ravichandiran ◽  
S. Karthi ◽  
R. Shrimathi ◽  
S. Adline Princy
Keyword(s):  
Virtual Screening ◽  
Quorum Sensing ◽  
E Coli ◽  
Melia Dubia

scholarly journals Quorum Sensing Signal Selectivity and the Potential for Interspecies Cross Talk

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Samantha Wellington ◽  
E. Peter Greenberg
Keyword(s):  
Quorum Sensing ◽  
Cell Signaling ◽  
Cross Talk ◽  
Homoserine Lactone ◽  
Interspecies Interactions ◽  
Interspecies Competition ◽  
Content Type ◽  
E Coli ◽  
Competition And Cooperation ◽  
Wide Range

ABSTRACTMany species of proteobacteria communicate with kin and coordinate group behaviors through a form of cell-cell signaling called acyl-homoserine lactone (AHL) quorum sensing (QS). Most AHL receptors are thought to be specific for their cognate signal, ensuring that bacteria cooperate and share resources only with closely related kin cells. Although specificity is considered fundamental to QS, there are reports of “promiscuous” receptors that respond broadly to nonself signals. These promiscuous responses expand the function of QS systems to include interspecies interactions and have been implicated in both interspecies competition and cooperation. Because bacteria are frequently members of polymicrobial communities, AHL cross talk between species could have profound impacts. To better understand the prevalence of QS promiscuity, we measured the activity of seven QS receptors in their native host organisms. To facilitate comparison of our results to previous studies, we also measured receptor activity using heterologous expression inEscherichia coli. We found that the standardE. colimethods consistently overestimate receptor promiscuity and sensitivity and that overexpression of the receptors is sufficient to account for the discrepancy between native andE. colireporters. Additionally, receptor overexpression resulted in AHL-independent activity inPseudomonas aeruginosa. Using our activation data, we developed a quantitative score of receptor selectivity. We find that the receptors display a wide range of selectivity and that most receptors respond sensitively and strongly to at least one nonself signal, suggesting a broad potential for cross talk between QS systems.IMPORTANCESpecific recognition of cognate signals is considered fundamental to cell signaling circuits as it creates fidelity in the communication system. In bacterial quorum sensing (QS), receptor specificity ensures that bacteria cooperate only with kin. There are examples, however, of QS receptors that respond promiscuously to multiple signals. “Eavesdropping” by these promiscuous receptors can be beneficial in both interspecies competition and cooperation. Despite their potential significance, we know little about the prevalence of promiscuous QS receptors. Further, many studies rely on methods requiring receptor overexpression, which is known to increase apparent promiscuity. By systematically studying QS receptors in their natural parent strains, we find that the receptors display a wide range of selectivity and that there is potential for significant cross talk between QS systems. Our results provide a basis for hypotheses about the evolution and function of promiscuous signal receptors and for predictions about interspecies interactions in complex microbial communities.

scholarly journals Homologous Quorum Sensing Regulatory Circuit: A Dual-Input Genetic Controller for Modulating Quorum Sensing-Mediated Protein Expression in E. coli

2020 ◽  
Vol 9 (10) ◽  
pp. 2692-2702
Author(s):  
Pricila Hauk ◽  
Kristina Stephens ◽  
Chelsea Virgile ◽  
Eric VanArsdale ◽  
Alex Eli Pottash ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Protein Expression ◽  
E Coli ◽  
Regulatory Circuit
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