Wang Hsinkai, Yang Ya-Tang
Department of Electrical Engineering, National Tsing Hua University , Hsinchu, 30013, Taiwan, R.O.C.
ACS Synth Biol. 2017 Sep 15;6(9):1793-1796. doi: 10.1021/acssynbio.7b00091. Epub 2017 Jun 5.
The current standard protocols for characterizing the optogenetic circuit of bacterial cells using flow cytometry in light tubes and light exposure of culture plates are tedious, labor-intensive, and cumbersome. In this work, we engineer a bioreactor with working volume of ∼10 mL for in vivo real-time optogenetic characterization of E. coli with a CcaS-CcaR light-sensing system. In the bioreactor, optical density measurements, reporter protein fluorescence detection, and light input stimuli are provided by four light-emitting diode sources and two photodetectors. Once calibrated, the device can cultivate microbial cells and record their growth and gene expression without human intervention. We measure gene expression during cell growth with different organic substrates (glucose, succinate, acetate, pyruvate) as carbon sources in minimal medium and demonstrate evolutionary tuning of the optogenetic circuit by serial dilution passages.
目前，使用流式细胞术在光管中对细菌细胞的光遗传学电路进行表征以及对培养板进行光照的标准方案既繁琐、劳动强度大又麻烦。在这项工作中，我们设计了一种工作体积约为10 mL的生物反应器，用于对具有CcaS-CcaR光传感系统的大肠杆菌进行体内实时光遗传学表征。在该生物反应器中，四个发光二极管光源和两个光电探测器可进行光密度测量、报告蛋白荧光检测以及光输入刺激。一旦校准，该设备就能在无需人工干预的情况下培养微生物细胞并记录其生长和基因表达。我们在基本培养基中使用不同有机底物（葡萄糖、琥珀酸盐、乙酸盐、丙酮酸盐）作为碳源培养细胞时测量基因表达，并通过连续稀释传代展示了光遗传学电路的进化调整。
