Fracassi Chiara, Postiglione Lorena, Fiore Gianfranco, di Bernardo Diego
TeleThon Institute of Genetics and Medicine (TIGEM) , 80078 Pozzuoli, Italy.
Department of Electrical Engineering and Information Technology, University of Naples Federico II , 80125 Naples, Italy.
ACS Synth Biol. 2016 Apr 15;5(4):296-302. doi: 10.1021/acssynbio.5b00141. Epub 2015 Oct 6.
Automatic control of gene expression in living cells is paramount importance to characterize both endogenous gene regulatory networks and synthetic circuits. In addition, such a technology can be used to maintain the expression of synthetic circuit components in an optimal range in order to ensure reliable performance. Here we present a microfluidics-based method to automatically control gene expression from the tetracycline-inducible promoter in mammalian cells in real time. Our approach is based on the negative-feedback control engineering paradigm. We validated our method in a monoclonal population of cells constitutively expressing a fluorescent reporter protein (d2EYFP) downstream of a minimal CMV promoter with seven tet-responsive operator motifs (CMV-TET). These cells also constitutively express the tetracycline transactivator protein (tTA). In cells grown in standard growth medium, tTA is able to bind the CMV-TET promoter, causing d2EYFP to be maximally expressed. Upon addition of tetracycline to the culture medium, tTA detaches from the CMV-TET promoter, thus preventing d2EYFP expression. We tested two different model-independent control algorithms (relay and proportional-integral (PI)) to force a monoclonal population of cells to express an intermediate level of d2EYFP equal to 50% of its maximum expression level for up to 3500 min. The control input is either tetracycline-rich or standard growth medium. We demonstrated that both the relay and PI controllers can regulate gene expression at the desired level, despite oscillations (dampened in the case of the PI controller) around the chosen set point.
对活细胞中的基因表达进行自动控制对于表征内源性基因调控网络和合成电路至关重要。此外，这种技术可用于将合成电路组件的表达维持在最佳范围内，以确保可靠的性能。在此，我们提出一种基于微流控的方法，用于实时自动控制哺乳动物细胞中四环素诱导型启动子的基因表达。我们的方法基于负反馈控制工程范式。我们在一个单克隆细胞群体中验证了我们的方法，该群体在具有七个四环素响应操作基序（CMV-TET）的最小CMV启动子下游组成性表达荧光报告蛋白（d2EYFP）。这些细胞还组成性表达四环素反式激活蛋白（tTA）。在标准生长培养基中生长的细胞中，tTA能够结合CMV-TET启动子，导致d2EYFP最大程度表达。向培养基中添加四环素后，tTA从CMV-TET启动子上脱离，从而阻止d2EYFP表达。我们测试了两种不同的与模型无关的控制算法（继电器和比例积分（PI）），以迫使一个单克隆细胞群体在长达3500分钟的时间内表达等于其最大表达水平50%的中间水平的d2EYFP。控制输入要么是富含四环素的培养基，要么是标准生长培养基。我们证明，尽管在选定设定点周围存在振荡（PI控制器的情况下振荡会减弱），但继电器和PI控制器都可以将基因表达调节到所需水平。
