Comparison and Calibration of Different Reporters for Quantitative Analysis of Gene Expression
Comparison and Calibration of Different Reporters for Quantitative Analysis of Gene Expression
复制标题
DOI：
10.1016/j.bpj.2011.06.026
复制
发表时间：
2011-08-03
影响因子：
3.4
通讯作者：
Phillips, Rob
中科院分区：
文献类型：
--
作者：
Garcia, Hernan G.;Lee, Heun Jin;Phillips, Rob
观察到细菌中基因表达的绝对水平变化多达六个数量级。热力学模型已被提出作为一种工具来描述一个给定的转录电路的表达水平。在这种情况下，它是必不可少的，以了解不同的方法来测量基因表达的局限性和线性范围，并确定在何种程度上测量从不同的报告可以直接比较的一个目标是严格测试的基因表达的理论描述。在这篇文章中，我们比较了两个蛋白质报告，通过测量的绝对水平的表达和倍数变化的表达，使用荧光蛋白EYFP和酶报告β-半乳糖苷酶。我们确定其动态和线性范围，并表明它们是可互换的测量平均水平的表达超过四个数量级。通过校准这些报告基因，使得它们可以用绝对分子计数来解释，我们建立了它们的适用性限制：EYFP表达低端的自发荧光（近似于每个细胞10个分子）和β-半乳糖苷酶表达高端的细胞生长干扰（近似于每个细胞20，000个分子）。这些品质使得报告者在试图通过实验验证理论模型的预测时是互补的和必要的。
Absolute levels of gene expression in bacteria are observed to vary over as much as six orders of magnitude. Thermodynamic models have been proposed as a tool to describe the expression levels of a given transcriptional circuit. In this context, it is essential to understand both the limitations and linear range of the different methods for measuring gene expression and to determine to what extent measurements from different reporters can be directly compared with one aim being the stringent testing of theoretical descriptions of gene expression. In this article, we compare two protein reporters by measuring both the absolute level of expression and fold-change in expression using the fluorescent protein EYFP and the enzymatic reporter beta-galactosidase. We determine their dynamic and linear range and show that they are interchangeable for measuring mean levels of expression over four orders of magnitude. By calibrating these reporters such that they can be interpreted in terms of absolute molecular counts, we establish limits for their applicability: autofluorescence on the lower end of expression for EYFP (at similar to 10 molecules per cell) and interference with cellular growth on the high end for beta-galactosidase (at similar to 20,000 molecules per cell). These qualities make the reporters complementary and necessary when trying to experimentally verify the predictions from the theoretical models.