[论文解读] Cell-cycle-synchronized, oscillatory expression of a negatively autoregulated gene in E. coli

Zach Hensel, Tatiana T. Marquez‐Lago|arXiv (Cornell University)|Jun 29, 2015

Gene Regulatory Network Analysis参考文献 42被引用 3

一句话总结

本研究证明，基于λ噬菌体Cro阻遏蛋白的染色体整合、负反馈自调节基因回路即使在缺乏协同性、快速降解或长延迟的情况下，也能在大肠杆菌中表现出稳健的、与细胞周期同步的振荡。振荡源于反馈驱动的动力学与依赖于细胞周期的基因复制的耦合，相位偏移与估算的基因加倍时间精确相关。

ABSTRACT

Engineering genetic networks to be both predictable and robust is a key challenge in synthetic biology. Synthetic circuits must reliably function in dynamic, stochastic and heterogeneous environments, and simple circuits can be studied to refine complex gene-regulation models. Although robust behaviours such as genetic oscillators have been designed and implemented in prokaryotic and eukaryotic organisms, a priori genetic engineering of even simple networks remains difficult, and many aspects of cell and molecular biology critical to engineering robust networks are still inadequately characterized. Particularly, periodic processes such as gene doubling and cell division are rarely considered in gene regulatory models, which may become more important as synthetic biologists utilize new tools for chromosome integration. We studied a chromosome-integrated, negative-feedback circuit based upon the bacteriophage λ transcriptional repressor Cro and observed strong, feedback-dependent oscillations in single-cell time traces. This finding was surprising due to a lack of cooperativity, long delays or fast protein degradation. We further show that oscillations are synchronized to the cell cycle by gene duplication, with phase shifts predictably correlating with estimated gene doubling times. Furthermore, we characterized the influence of negative feedback on the magnitude and dynamics of noise in gene expression. Our results show that cell-cycle effects must be accounted for in accurate, predictive models for even simple gene circuits. Cell-cycle-periodic expression of λ Cro also suggests an explanation for cell-size dependence in lysis probability and an evolutionary basis for site-specific λ integration.

研究动机与目标

• 研究在生理条件下，一个最小化的、负反馈自调节的基因回路是否能在大肠杆菌中产生稳健的振荡。
• 考察细胞周期动力学（尤其是基因复制）在同步化和塑造合成基因回路振荡行为中的作用。
• 量化负反馈对单细胞基因表达中噪声幅度和动力学的影响。
• 评估与细胞周期同步的表达对λ噬菌体裂解时机及整合进化约束的影响。

提出的方法

• 构建了一个以λ噬菌体Cro阻遏蛋白作为转录阻遏蛋白的合成基因回路，并将其整合到大肠杆菌染色体中。
• 利用时间序列荧光显微成像技术实时监测单细胞中Cro基因的表达动力学。
• 采用随机微分方程对系统进行建模，以捕捉内在噪声和反馈动力学。
• 通过追踪细胞分裂和基因复制事件，将振荡相位与细胞周期进程相关联。
• 改变启动子强度和阻遏蛋白降解速率等参数，以评估系统的鲁棒性。
• 分析振荡相位相对于细胞周期时间的偏移，以推断基因加倍动力学。

实验结果

研究问题

• RQ1 一个简单、非协同性、负反馈自调节的基因回路是否能在大肠杆菌中产生持续振荡？
• RQ2 细胞周期进程（尤其是基因复制）如何影响振荡的时机与同步性？
• RQ3 负反馈在多大程度上减少了细胞周期中基因表达的噪声？
• RQ4 为何λ噬菌体特异性地整合到大肠杆菌染色体中？振荡性表达与此有何关联？

主要发现

• 尽管缺乏协同性或快速降解，负反馈自调节的Cro回路在单细胞时间轨迹中仍表现出强烈且依赖反馈的振荡。
• 振荡与细胞周期同步，相位偏移与估算的基因加倍时间具有可预测的相关性。
• 该系统对内在噪声表现出鲁棒性，负反馈显著降低了表达变异性。
• Cro的周期性细胞周期表达为λ噬菌体裂解概率的细胞大小依赖性提供了机制解释。
• 研究结果提示了位点特异性λ整合的进化基础，因为振荡性表达可能通过与细胞周期协调而得到优化。
• 结果表明，在设计合成基因回路时，必须显式建模细胞周期效应，才能实现准确且可预测的设计。