Difference between revisions of "Team:SDSZ China/Mo"

Bacterial cells produce and release small amounts of chemical signaling molecules referred to as autoinducers, e.g. N-Acyl Homoserine Lactones (AHL) found in gram-negative bacteria.And bacterial quorum sensing is a form of cellular communication mediated by autoinducers. 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 the 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 those for autoinducer 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 synchronizes 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.

Our model is based on a synthetic lux system as a tunable cell density sensor-regulator which have been previously researched. Autoinduction of luminescence in the marine bacteria V. fischeri and Vibrioharveyi was described in the early 1970s. When these bacteriaare cultured in broth,they exhibit a lag in luminescence gene (lux)expression during early and mid-exponential growth, followed by a rapid increase inexpression 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 ens- uring 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 the response to reaching appropriate cell density, self-trigger the expression of the appropriate pathway genes for efficient target compound production.

Synthetic lux promoter PluxlacO activity can be depending on exogenous IPTG concentration 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. This inhibition is reduced depending on the Figure 2. Design of the lux system upon which we built up the model and conducted our research concentration of exogenous IPTG. 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. 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.

There has been a great number of work conducted on the quorum sensing systems. Many experimental or modeling studies focus on biofilm growth, dispersal or quorum sensing induction. And many different types of models of quorum sensing have been built-up applying to various conditions. For examples, there are single-cell model, population model, One-Population Model with Mass Transfer: QS Versus DS, The Discrete Capsule Model, The Continuum Colony Mode and even more. We aimed to develop a model upon the MTS, by which microbial self-induced metabolic state is able to switch from cell growth to isopropanol production. Based on our team’s knowledge, there was not any experimental researches on the plasmid pTA1083 and pTA1109, the components in this particular lux system. Thus, regarding on our experiment, it is necessary to build up a model upon MTS to confirm its usefulness, with the help of our experiment data and previous studies as reference.

Since we were not able to derive a mathematic formula based on our current knowledge, we found several correlations out of the data. As the the concentration of the IPTG(between 0.01mM and 1.0mM) gets higher, the concentration of glucose increases, and the concentration of isopropanol and cell density goes lower. However there are exceptions out of this pattern due to instrumental errors or unclear reasons.

Population Model of Quorum Sensing with Multiple Parallel Pathways
Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production
A Mathematical Model of Quorum Sensing Induced Biofilm Detachment
Combination Therapy Strategy of Quorum Quenching Enzyme and Quorum Sensing Inhibitor in Suppressing Multiple Quorum Sensing Pathways of P. aeruginosa
Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli
Quorum Sensing in Bacteria: the LuxR-LuxI Family of Cell Density-Responsive Transcriptional Regulatorst
Inhibition of Lux quorum-sensing system by synthetic N-acyl-L-homoserine lactone analogous.