Environmentally-modulated changes in fluorescence distribution in cells with oscillatory genetic network dynamics.
Environmentally-modulated changes in fluorescence distribution in cells with oscillatory genetic network dynamics.
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DOI：
10.1016/j.jbiotec.2009.01.011
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发表时间：
2009-03-25
影响因子：
4.1
通讯作者：
Mantzaris N
中科院分区：
文献类型：
--
作者：
Portle S;Iadevaia S;San KY;Bennett GN;Mantzaris N
我们研究了表达人工基因调控网络（称为“抑制器”）的大肠杆菌细胞群中绿色荧光蛋白（GFP）表达水平的分布，该网络在单细胞水平上表现出振荡。使用流式细胞术进行和分析了一系列摇瓶实验，以测试如何使用诱导剂脱水四环素（aTc）和异丙基-β-D-硫代半乳糖苷（IPTG）在细胞外控制携带该系统的细胞群。随着 [aTc] 的变化，它表现出双阈值行为，使得整个文化达到准时间不变“参考状态”的三个稳定状态之一。此外，还存在显着的滞后现象。瞬态中，中间状态显示阻尼振荡，而低状态和高状态显示稳定稳态。 IPTG 的添加用于微调仅 aTc 表达的特征，降低分布的平均值和 CV，并可能将网络扰乱到不同的状态。然而，在对该系统进行建模时，根据设计的交互，多重性和双阈值行为在理论上是不可能的。为了解释这种差异，我们假设一个或多个阻遏蛋白与不包含其操纵位点的启动子具有显着的非特异性相互作用。包含这些额外相互作用的新建模结果在质量上与我们的实验结果相符。在构建质粒来测试这些假设后，我们发现至少存在四种相互作用，它们可以产生实验中看到的低态和高态以及多重性。这种遗传结构的行为具有前所未有的灵活性，实验和建模的结合启发了我们对驱动网络行为的分子相互作用的理解，使我们发现非特异性相互作用的重要性。
We investigated the distribution of green fluorescent protein (GFP) expression levels in a population of E. coli cells expressing an artificial genetic regulatory network, known as the “repressilator”, which exhibits oscillations at the single-cell level. A series of shake flask experiments were performed and analyzed using flow cytometry to test how cell populations carrying this system could be controlled extracellularly using the inducers anhydrotetracycline (aTc) and isopropyl-β-D-thiogalactopyranoside (IPTG). With variation of [aTc], it exhibits bi-threshold behavior, such that the entire culture reaches one of three steady states at a quasi-time invariant “reference state.” Also, there is significant hysteresis. Transiently, the middle state shows damping oscillations, while the low and high states show a stable steady state. The addition of IPTG serves to fine-tune the characteristics of the aTc-only expression, lowering the average and CV of the distributions, and possibly perturbing the network to a different state. However, in modeling this system, the multiplicity and bi-threshold behavior are not theoretically possible according to the designed interactions. In order to explain this discrepancy, we hypothesize that one or more of the repressors has a significant nonspecific interaction with a promoter that does not contain its operator site. The new modeling results incorporating these extra interactions qualitatively match our experimental findings. After constructing plasmids to test these hypotheses, we discover that at least four of these interactions exist, which can create the low and high states and multiplicity seen experimentally. This genetic architecture has flexibility in its behavior that has not been demonstrated before, and the combination of experiment and modeling enlightened our understanding of the molecular interactions driving the network's behavior, leading us to discover the significance of nonspecific interactions.
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影响因子：
64.8
作者：
Becskei, A;Serrano, L
通讯作者：
Serrano, L
DOI：
10.1073/pnas.74.10.4228
发表时间：
1977-01-01
影响因子：
11.1
作者：
KAOHUANG, Y;REVZIN, A;VONHIPPEL, PH
通讯作者：
VONHIPPEL, PH
DOI：
10.1073/pnas.43.7.553
发表时间：
1957-01-01
影响因子：
11.1
作者：
NOVICK, A;WEINER, M
通讯作者：
WEINER, M
DOI：
10.1016/0025-5564(67)90008-9
发表时间：
1967-01-01
期刊：
MATH BIO SCL
影响因子：
--
作者：
FREDERICKSON, A. G.;RAMKRISHNA, D.;TSUCHIYA, H. M.
通讯作者：
TSUCHIYA, H. M.
影响因子：
14.9
作者：
Lutz, R;Bujard, H
通讯作者：
Bujard, H