Mathematical Modeling And Stability Of Suicide Gene Loop Based On Bacterial Population Induction

The suicidal behavior of bacteria is a common phenomenon in nature, which is the mechanism of programmed cell death in procaryotic organism. Quorum sensing(QS) is a form of communication between the bacterial cells. When the signal molecule synthesized and secreted by cells reaches a certain threshold, it will activate a series of genes, and this will synchronize bacteria species to realize some functions and changes in behavior. Suicide gene based on quorum sensing circuit has been built, which have a great significance both in theory and application.This paper uses the thought of synthetic biology to research circuits based on Lux R-Lux I system. It combines mathematical model and molecular biology experiments to simulate the growth of population and suicidal behavior. The purpose is to achieve long-term, stable and controllable system through analysis IPTG concentration, p H, temperature and nutritional condition. This paper also discusses deeply the influence of nutrition conditions on Plux promoter. It is discovered that two upstream regulation sequence have effects on the promoter, which deepen the understanding of bacterial quorum sensing.The genetic circuit of p Popctrl plasmid can secrete a kind of homoserine signaling molecules(acyl- homoserine lactone, AHL) in the presence of IPTG in Escherichia coli. AHL could accumulate to a certain threshold to combine the transcription factor of Lux R and activate the Lux R to Lux R*. Lux R* promote the Plux to express the Ccd B protein to kill the cell. The growth and the suicidal behavior of bacteria were studied through the mathematical model based on the differential equation. The model is consistent with experimental data when measuring the colony forming unit(CFU) at different time points through changing the IPTG concentration, p H, temperature and nutrition conditions.This study demonstrated that, when the temperature and the p H value keep invariant, IPTG concentration has a positive effect on Plac promoter to activate AHL production mediated by Lux I. The greater the IPTG value, the more obvious the amount of suicidal bacteria through activating the Ccd B protein. When the temperature and IPTG concentration keep invariant, the p H value affects the degradation of AHL which is easily degraded in alkaline environment. Therefore, the lower the p H, the more obvious the amount of suicidal bacteria through activating the Ccd B protein. When IPTG concentration and p H value keep invariant, the temperature positively affect bacterial specific growth rate and Lux R*. Specific growth rate is slow and Lux R* protein are stable, the phemomenon of bacterial suicide behavior is obvious at 30℃. When changing the condition of the medium, the bacterial suicide oscillation circuit disappear in M9 medium, which is the internal mechanism at molecular level. Finally, the effective combination of experiment and mathematical model is one of the effective methods to guide experiments by mathematical model.The nutritional condition has an important influence on genetic circuits and suicidal behavior of bacteria. In M9 medium, there is no suicidal phenomenon, which means that QS has potential and unknown regulatory mechanism related with nutrition. GFP fluorescence gene was connected to the downstream of QS system, which is more intuitive to observe the regulation mechanism of QS system rapidly. The experiment founded that, in addition to the QS system, other regulatory mechanisms also influence the expression of GFP fluorescence. Through a series of experimental analysis, the paper founded that wild type promoter Plux is weak constitutive promoter, which has the background level. Its expression level can almost be ignored and has to depend on the regulation of transcription factors. Promoter Plux is regulated by luxbox sequences and crp sequences of transcriptional regulation factor. Under common circumstances, Plux promoter is regulated by luxbox sequences related lux R/I system, according to the law of regulation by QS system. But when lacking carbon source or nutrition, c AMP and CRP protein can form c AMP-CRP, the compound could combine crp sequences to promote Plux expression, but if there are AHLs, the presence of crp sequences will suppress promoter Plux expression combined with luxbox sequences and its background level expression.