Automatic Control of Gene Expression in Mammalian Cells
Automatic Control of Gene Expression in Mammalian Cells
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DOI：
10.1021/acssynbio.5b00141
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发表时间：
2016-04-01
影响因子：
4.7
通讯作者：
di Bernardo, Diego
中科院分区：
文献类型：
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作者：
Fracassi, Chiara;Postiglione, Lorena;di Bernardo, Diego
活细胞中基因表达的自动控制对于表征内源基因调控网络和合成回路都是至关重要的。此外，这种技术可以用于将合成电路元件的表达保持在最佳范围内，以确保可靠的性能。在这里，我们提出了一种基于微流体的方法，可以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，并进行真实的实时控制。我们的方法是基于负反馈控制工程范式。我们验证了我们的方法在单克隆细胞群体组成型表达的荧光报告蛋白（d2 EYFP）的最小CMV启动子下游与七个tet响应的操作员基序（CMV-TET）。这些细胞也组成型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d2 EYFP最大化表达。在向培养基中加入四环素后，tTA从CMV-TET启动子上分离，从而阻止d2 EYFP表达。我们测试了两种不同的模型独立的控制算法（继电器和比例积分（PI）），迫使单克隆细胞群表达的中间水平的d2 EYFP等于其最大表达水平的50%，长达3500分钟。控制输入是富含四环素或标准生长培养基。我们证明了继电器和PI控制器都可以在所需的水平上调节基因表达，尽管在所选的设定点周围存在振荡（在PI控制器的情况下被抑制）。
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 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.