| In recent years,designs and constructions of different genetic circuits and synthetic systems have been received more attentions in synthetic biology research.This research field has important application values in environmental governance,human health and industrial production.Development of tools and biological parts is essential for the construction of devices.Quorum sensing(QS)is a mechanism for bacteria to regulate gene expression and coordinate population behavior according to the changes in cell population density.It is also an intercellular communication tool which is frequently used in synthetic biology research.However,the ubiquitous crosstalk among QS systems limits the construction of complex circuits and systems.In the research of the orthogonality in LuxI/R type QS systems,the effect of acylhomoserine lactone(AHL)synthase on signal orthogonality is often ignored,resulting in the limitation of the applications with orthogonal QS systems.Constructing different genetic circuits is one of the major research directions in synthetic biology.It is important to realize the sequential gene expression in an automatic delayed cascade control.The design and exploration of microbial consortia is an emerging research field in the construction of synthetic systems.However,there are many uncontrollable and immeasurable factors in nature.The controllable and uncomplicated characterizations of synthetic microbial ecosystems are important for the further study and applications of microbial consortia.In this study,completely orthogonal QS systems were established in Escherichia coli with the intracellular expression of AHL synthase.The orthogonal QS systems were applied to design genetic circuits,resulting in the construction of an automatic cascade circuit.Furthermore,the synthetic competitive and symbiotic ecosystems were constructed based on the completely orthogonal QS systems,in which the population proportion could be regulated.1.Establishment of completely orthogonal QS systems and the application in genetic circuit constructionBy analyzing the orthogonality and crosstalk between LuxI/R-type QS systems,the Tra system from Agrobacterium tumefaciens and the Las system from Pseudomonas aeruginosa were chosen to construct the completely orthogonal QS systems.The Las and Tra systems are promoter orthogonal and signal crosstalk with each other.In order to achieve signal orthogonality,we analyzed the sensitivity of receptors to different signals.We found that the sensitivity of LasR to 3OC6HSL was much lower than that to 3OC8HSL,while the sensitivities of TraR to 3OC6HSL and 3OC8HSL were similar.Therefore,we selected EsaI from Esa system to synthesize 3OC6HSL as the autoinducer of the Tra system.Compared to the Tra system's homologous signal 3OC8HSL,3OC6HSL can reduce the signal crosstalk between two systems.As the Tra system exhibits poor activity in E.coli,we improve the expression of the Tra system by substituting Ptra and TraR with modified Ptra*and TraR(W),and the recombinant system was named as Tra*system.L-arabinose(Ara)and anhydrotetracycline(aTc)were used to induce EsaI and LasI to synthesize 3OC6HSL and 3OC12HSL,respectively.Two expression plasmids of the Tra*and the Las systems were constructed in E.coli TOP 10.After initial characterization,both systems have serious leaked expression.Further characterizations suggested that the leakage of both systems was caused by the leaky production of signal molecules.Leakage of two systems were reduced by modifying the Ribosomal Binding Site(RBS)and promoter of AHL synthase.Finally,ideal modified systems and plasmids were obtained.Then,we verified the orthogonality between systems.Basing on two expression plasmids,a series of verification plasmids were constructed by deleting receptors or/and QS promoters.These plasmids were transferred into E.coli TOP 10 in pairs.Different inducers were added to induce the expression of the corresponding systems.Orthogonality of systems was indicated by the expression of reporters.Verification results suggested that there was a signal crosstalk between two systems.This crosstalk was caused by the nonhomologous AHL produced by EsaI.To solve this problem,a library of LasR mutants was constructed by random mutation.Two ideal mutants with reduced sensitivity to nonhomologous signals were screened by GFP fluorescence with the addition of 3OC12HSL.Both mutants eliminated the signal crosstalk.The mutation of LasR also reduced its sensitivity to the homologous signal 3OC12HSL.The production of 3OC12HSL was further increased.After optimization the expression of the Las system,the complete orthogonality between two systems was finally realized.The completely orthogonal QS systems were used to construct a genetic circuit.An automatic cascade circuit was constructed to realize the sequential expression of target genes.The function of cascade circuit was verified at both translation and transcription levels.2.Construction of synthetic ecosystems based on orthogonal QS systems that can regulate the population proportionIn the design of the synthetic ecosystems,the completely orthogonal QS systems were applied as communication module,and the CcdA/B toxin-antitoxin system was used as effect module.Strains CG and CR were designed in the synthetic competitive ecosystem.Both strains contain a positive feedback causing self-rescue through the cis-acting AHL activation of antitoxin and a negative feedback causing the population limitation through the trans-acting AHL activation of toxin.Strains SG and SR were designed in the synthetic symbiotic ecosystem.Both strains contain a negative feedback causing self-limitation through the cis-acting AHL activation of toxin and a positive feedback causing the population rescue through the trans-acting AHL activation of antitoxin.The transformation of different ecological relationships was realized simply by reconfiguration of the module's layout.By regulating the expression of key genes with different RBS,several strains were constructed.They were characterized with different inducers and the ideal strains were selected according to the growth.The plate co-culture experiment was conducted to visualize two ecological relationships,and the competitive and symbiotic interactions between strains were observed.It was found that regulating the expression of key genes in the competitive ecosystem realized the exchange of dominant strains.In the symbiotic ecosystem,different culture medium altered the symbiotic relationship.Therefore,both internal expression regulation and external culture environment could change the interaction between strains.Each strain owned two independent feedback control circuits in two synthetic ecosystems.This design makes the positive and negative growth regulation was independent with each other.Meanwhile,QS system is an induction system that responds to population density.Therefore,these ecosystems have the potential to regulate population proportion.On the basis of two synthetic ecosystems,two inducible synthetic ecosystems which could regulate the subpopulation proportion were constructed by further modification.In inducible synthetic ecosystems,the QS promoters of AHL synthases were replaced with inducible promoters induced by Ara or aTc.The inducers were used to control the synthesis of AHL,and then the expression of effect module was regulated,so as to regulate the population proportion.By adding different inducers to the co-culture systems,a large range of proportional regulation between subpopulations was successfully achieved.In summary,we constructed a pair of completely orthogonal QS systems,which extended the toolbox of synthetic biology research.The completely orthogonal QS systems were used to construct a cascade circuit which realized the autonomous sequential expression of genes.The completely orthogonal QS systems were also applied in the construction of synthetic competitive and symbiotic ecosystems,which realized the regulation of subpopulation ratio. |
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