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− | + | Bacteria produce and release small amounts of chemical signaling molecules known as auto-inducers [e.g. N-Acyl Homoserine Lactones (AHL)] found in gram-negative bacteria. And bacterial quorum sensing is a form of cellular communication mediated by auto-inducers. Quorum sensing allows populations of bacteria to track their cell density and possibly other environmental factors, and use this as a basis for synchronizing gene expression at certain population level. Each cell also has receptors that can specifically detect the signaling molecule via ligand–receptor binding, which then activates transcription of certain genes, including the transcription for auto-inducer synthesis. The probability of signal detection will depend on the local extracellular concentration of autoinducers, which itself will depend on cell density. Hence, higher cell densities will tend to generate higher concentrations of autoinducer and thus increase the likelihood of binding to receptors and the activation of downstream gene networks. Once a threshold environmental autoinducer concentration level is reached, the bacteria undergo alterations in gene expression which synchronize collective behavior. This up-regulation of cells typically also invokes signal production at an increased rate. Acylhomoserine lactone (AHL)-based QS signals are found in more than 70 bacterial species, in which many of them are pathogens. | |
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− | Our model is based on a synthetic lux system as a tunable cell density sensor-regulator which | + | Our model is based on a synthetic lux system as a tunable cell density sensor-regulator which has been previously researched. Autoinduction of luminescence in the marine bacteria V. fischeri and Vibrioharveyi was described in the early 1970s. When these bacteria are cultured in broth, they exhibit a lag in luminescence gene (lux) expression during early and mid-exponential growth, followed by a rapid increase in expression during the late exponential and early stationary growth phases. In this design, accessibility of QS-signal (AHL–LuxR complex) to PluxlacO is interfered by LacI repressor expressed from the constitutive PlacIq promoter at attB locus of chromosome. |
− | This system has formed a positive feedback after reaching the threshold. And a synthetic circuit is called Metabolic toggle switch (MTS) controls conditional redirection of metabolic flux from endogenous pathways toward targeted synthetic metabolic pathway.This flux regulation system significantly improved the isopropanol production of an engineered Escherichia coli strain by drastically switching the metabolic flux from cell mass development to production of the target compound. It required strict timing of the addition of exogenous chemical inducer in order to achieve its desired function of | + | This system has formed a positive feedback after reaching the threshold. And a synthetic circuit is called Metabolic toggle switch (MTS) controls conditional redirection of metabolic flux from endogenous pathways toward targeted synthetic metabolic pathway. This flux regulation system significantly improved the isopropanol production of an engineered Escherichia coli strain by drastically switching the metabolic flux from cell mass development to production of the target compound. It required strict timing of the addition of exogenous chemical inducer in order to achieve its desired function of ensuring adequate cell mass and improvement in productivity of the target compound. To decrease such difficulty, ideally, the engineered microbes would sense their population, and in response to reaching appropriate cell density, self-trigger the expression of the appropriate pathway genes for efficient target compound production.</p> |
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− | Synthetic lux promoter PluxlacO activity | + | Synthetic lux promoter PluxlacO activity depends on exogenous IPTG concentration, with IPTG added at the beginning of the fermentation. The PluxlacO promoter is designed so the AHL-LuxR complex mediated transcription activity is inhibited by the LacI repressor. |
− | + | The lux system-mediated detection of cell density requires the interaction of a diffusible auto-inducer acylhomoserine lactone (AHL) and AHL-dependent transcription activator LuxR. This design enables the QS system to prevent the leaky background expression from lux promoter through inhibition by LacI repressor. Additionally, the transcription activity of this promoter could be tuned because the repression intensity of LacI repressor is modulated by interaction with the inducer, IPTG. The plasmid pTA1083 was used as the QS signal receptor/reporter module with yemGFP under the control of the synthetic lux promoter PluxlacO to evaluate the synthetic lux system. The pTA1109 plasmid was constructed as a QS signal generator module harboring luxI and luxR. | |
− | The lux system-mediated detection of cell density requires the interaction of a diffusible auto-inducer acylhomoserine lactone (AHL) and AHL-dependent transcription activator LuxR. This design enables the QS system to prevent the leaky background expression from lux promoter through inhibition by LacI repressor. Additionally, the transcription activity of this promoter could be tuned because the repression intensity of LacI repressor is modulated by interaction with the inducer, IPTG.The plasmid pTA1083 was used as the QS signal receptor/reporter module with yemGFP under the control of the synthetic lux promoter PluxlacO to evaluate the synthetic lux system.The pTA1109 plasmid was constructed as a QS signal generator module harboring luxI and luxR. | + | |
Additionally, this system triggers a metabolic toggle switch (MTS) in order to demonstrate the self- induced redirection of metabolic flux from the TCA cycle toward isopropanol production at a desired E. coli density. Isopropanol is one of the simplest secondary alcohols that can be dehydrated to yield propylene, a monomer for making polypropylene currently derived from petroleum. | Additionally, this system triggers a metabolic toggle switch (MTS) in order to demonstrate the self- induced redirection of metabolic flux from the TCA cycle toward isopropanol production at a desired E. coli density. Isopropanol is one of the simplest secondary alcohols that can be dehydrated to yield propylene, a monomer for making polypropylene currently derived from petroleum. | ||
Use of this metabolic toggle switch demonstrated a novel approach for intentionally switching intracellular metabolism as appropriate, from bacterial growth phase to bio-production phase, by direct regulation of specific metabolic flux. This is a general strategy which could be applied to other bio-productions in order to enhance metabolic flux to synthetic metabolic pathways while minimizing negative effects on bacterial growth and cell maintenance.</p> | Use of this metabolic toggle switch demonstrated a novel approach for intentionally switching intracellular metabolism as appropriate, from bacterial growth phase to bio-production phase, by direct regulation of specific metabolic flux. This is a general strategy which could be applied to other bio-productions in order to enhance metabolic flux to synthetic metabolic pathways while minimizing negative effects on bacterial growth and cell maintenance.</p> |
Latest revision as of 01:23, 18 October 2018