Hysteretic properties of a modular positive feedback-based gene amplifier

Synthetic Biology;positive feedback;hysteresis;cellular memory;dilution ratio

Poisson, Phillip J. ; Bhalerao ; Kaustubh

关键词: Synthetic Biology;    positive feedback;    hysteresis;    cellular memory;    dilution ratio;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/24292/Poisson_Phillip.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Positive feedback is a common mechanism in genetic circuits and can be used to achieve amplification, bistability, and hysteresis. Positive feedback mechanisms have been employed in numerous synthetic biology applications, including in the development of a modular positive feedback-based gene amplifier by Nistala et al. [1]. The modular design potentially enables use as a component in more complex synthetic gene networks, thus helping to achieve the oft-stated goal of designing well-characterized components that can be used in diverse synthetic biology applications. The positive feedback-based gene amplifier provides amplification of the maximum expression level of a gene product, and increased sensitivity to the inducer. However, the initial analysis by Nistala et al. did not demonstrate that this particular positive feedback mechanism can produce a hysteretic response to an inducer, i.e. exhibit memory of a prior stimulus. Positive feedback in a genetic circuit potentially, but not necessarily, leads to bistability, and a bistable circuit will produce some degree of hysteresis [2]. Therefore, the purpose of this research was to determine under what conditions, if any, the modular positive feedback-based gene amplifier will produce a hysteretic response. A modular gene amplifier with well-characterized hysteretic properties will potentially be a useful asset for synthetic biologists designing more complex networks out of simpler components. A simple mathematical model of gene expression in a positive-feedback system is used predict the conditions for bistability and hysteresis versus a simple graded response. Experiments are performed on the positive feedback-based tetracycline sensor in E. coli using green fluorescent protein (GFP) to measure expression level of the gene product. In the experiments, cells are placed in solutions of various inducer concentrations and grown to a steady-state level of GFP expression. Cells are then resuspended in solutions of lower inducer concentrations and allowed to reach steady state. Hysteresis is observed if the steady-state GFP level depends on the prior induction level of the cell culture. This research shows that the modular positive feedback-based gene amplifier is capable of producing a hysteretic response to a stimulus. The magnitude of the hysteretic response depended on the dilution ratio used to inoculate the cultures. Additionally, the magnitude of the hysteretic response depended partially on the final induction level of the culture, as higher induction levels resulted in a larger proportion of cells exhibiting a high level of GFP fluorescence. The cultures with induction history exhibited bistable GFP expression at all inducer concentrations. Cells exhibiting low GFP fluorescence grew at a faster rate than cells exhibiting high GFP fluorescence. This suggests that low dilution ratios provide more time for cells expressing at a low level to grow before the culture is saturated, and therefore make up a larger proportion of the final culture producing a smaller hysteretic response. These results provide a deeper understanding of the properties of the modular positive feedback-based gene amplifier and the conditions for hysteresis.