Reply to 'Complexity of molecular crowding in cell-free enzymatic reaction networks'.
Reply to 'Complexity of molecular crowding in cell-free enzymatic reaction networks'.
复制标题
回复“无细胞酶反应网络中分子拥挤的复杂性”。
DOI：
10.1038/nnano.2014.111
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发表时间：
2014
影响因子：
38.3
通讯作者：
LeDuc,Philip
中科院分区：
文献类型：
--
作者：
Tan,Cheemeng;Saurabh,Saumya;Bruchez,MarcelP;Schwartz,Russell;LeDuc,Philip
NATURE Nanotechnology|第9卷|六月2014| www.自然com/naturenanotechnology 407对应线（图1b）。图1b说明了Tan等人使用的实验方法的缺点。通过在1小时后取单个值来确定速率。当我们模拟pT 7 weak实验（参考文献3中的图2d）时，采用低20倍的RNA聚合酶的结合常数，拥挤对基因表达的影响似乎更大，尽管拥挤溶液的结合常数和速率常数的增加是相同的（图1c）。表观速率错误地表明，当在1小时后进行测量时，拥挤引起蛋白质表达的平台期，或者当在2小时后进行测量时引起双相反应，而实际上，溶液耗尽了资源，并且许多mRNA不能翻译成蛋白质。T7溶菌酶的共表达，如参考文献3的图3c所示，引入了负反馈回路。我们调整我们的模型，包括这个循环，假设拥挤的影响溶菌酶结合常数以相同的方式作为其他结合常数。我们的计算结果是惊人的（图1d）：如果可用的资源是无限的，拥挤不会导致双相反应，如参考文献3的图3d中难以令人信服的拟合所示。相反，溶菌酶的引入导致在所有拥挤密度下降低的有效青色荧光蛋白（CFP）生产速率。然而，如果资源有限，我们确实看到了一个明显的“双相”反应与日益拥挤。添加更高浓度的溶菌酶质粒使曲线向更低的有效CFP产生速率移动，这与预期一致，因为RNA聚合酶的增加部分在低活性状态下结合到溶菌酶10。上述对于区室化的无细胞基因表达系统意味着什么？原则上，没有理由说明为什么分子拥挤会对较大体积中的基因表达产生比小体积中更大的影响，并且更有可能的是其他效应（例如吸附到界面）导致DNA和/或蛋白质机器不再可用于基因表达。我们必须考虑到高度拥挤的环境的影响，并学会如何维持一个复杂的网络在一个囊泡内远离平衡地运行，在囊泡内资源将很快成为一个限制因素。
NATURE NANOTECHNOLOGY| VOL 9| JUNE 2014| www. nature. com/naturenanotechnology 407 correspondence lines in Fig. 1b). Figure 1b illustrates the shortcoming of the experimental method used by Tan et al. to determine rates by taking a single value after 1 hour. When we mimic the pT7weak experiment (Fig. 2d in ref. 3) by taking a 20-fold lower binding constant of RNA polymerase, there appears a larger apparent effect of crowding on gene expression, even though the increase in binding and rate constants for crowded solutions is the same (Fig. 1c). The apparent rates wrongly suggest that crowding gives rise to a plateau in protein expression when the measurement is taken after 1 hour, or a biphasic response when taken after 2 hours, whereas in reality the solution is depleted of resources and much of the mRNA cannot be translated into proteins. The co-expression of a T7 lysozyme, shown in Fig. 3c of ref. 3, introduces a negative feedback loop. We adapted our model to include this loop, assuming that crowding affects the lysozyme binding constants in the same way as other binding constants. The results of our calculation are striking (Fig. 1d): if the available resources are unlimited crowding does not lead to the biphasic response suggested by the hardly convincing fit in Fig. 3d of ref. 3. Instead, the introduction of lysozyme leads to a lowered effective cyan fluorescent protein (CFP) production rate at all crowding densities. However, if resources are limited we do see a clear ‘biphasic’response with increasing crowding. Adding higher concentrations of lysozyme plasmids shifts the curve to lower effective CFP production rates in agreement with expectations, as an increasing fraction of the RNA polymerase is bound to lysozyme in a state of low activity10. What does the above mean for compartmentalized cell-free gene expression systems? In principle, there is no reason why molecular crowding would exert a larger impact on gene expression in larger volumes than in small volumes, and it is much more likely that other effects such as adsorption to interfaces result in DNA and/or protein machinery no longer being available for gene expression.To conclude, it is becoming increasingly clear that to develop a synthetic cell, we must take into account the effect of the highly crowded environment and learn how to maintain a complex network operating far from equilibrium inside a vesicle where resources will quickly become a limiting factor.❐