The State Key Laboratory for Artificial Microstructures and Mesoscopic Physics School of Physics, Peking University, Beijing100871, China.
School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing100871, China.
ACS Synth Biol. 2022 Dec 16;11(12):3954-3965. doi: 10.1021/acssynbio.2c00307. Epub 2022 Oct 25.
is a common chassis for synthetic gene circuit studies. In addition to the dose-response of synthetic gene circuits, the analysis of dynamic responses is also an important part of the future design of more complicated synthetic systems. Recently, microfluidic-based methods have been widely used for the analysis of gene expression dynamics. Here, we established a two-layered microfluidic platform for the systematic characterization of synthetic gene circuits (eight strains in eight different culture environments could be observed simultaneously with a 5 min time resolution). With this platform, both dose responses and dynamic responses with a high temporal resolution could be easily derived for further analysis. A controlled environment ensures the stability of the bacterial growth rate, excluding changes in gene expression dynamics caused by changes of the growth dilution rate. The precise environmental switch and automatic micrograph shooting ensured that there was nearly no time lag between the inducer addition and the data recording. We studied four four-node incoherent-feedforward-loop (IFFL) networks with different operators using this device. The experimental results showed that as the effect of inhibition increased, two of the IFFL networks generated pulselike dynamic gene expressions in the range of the inducer concentrations, which was different from the dynamics of the two other circuits with only a simple pattern of rising to the platform. Through fitting the dose-response curves and the dynamic response curves, corresponding parameters were derived and introduced to a simple model that could qualitatively explain the generation of pulse dynamics.
是合成基因电路研究的常用底盘。除了合成基因电路的剂量反应外，动态响应的分析也是未来更复杂合成系统设计的重要组成部分。最近，基于微流控的方法已广泛用于基因表达动力学的分析。在这里，我们建立了一个双层微流控平台，用于系统地表征合成基因电路（可以同时观察八个不同培养环境中的八个菌株，具有 5 分钟的时间分辨率）。使用这个平台，可以轻松地获得具有高时间分辨率的剂量响应和动态响应，以便进一步分析。受控环境可确保细菌生长速率的稳定性，排除了由于生长稀释率变化而引起的基因表达动力学变化。精确的环境切换和自动显微拍摄确保了在诱导剂添加和数据记录之间几乎没有时间延迟。我们使用该设备研究了四个具有不同算子的四节点非相干前馈环（IFFL）网络。实验结果表明，随着抑制作用的增强，两个 IFFL 网络在诱导剂浓度范围内产生脉冲状的动态基因表达，这与另外两个只有简单上升到平台模式的电路的动力学不同。通过拟合剂量反应曲线和动态响应曲线，得出了相应的参数，并引入到一个简单的模型中，可以定性地解释脉冲动力学的产生。
