Vaccari Nicolás A, Zevallos-Aliaga Dahlin, Peeters Tom, Guerra Daniel G
Laboratorio de Moléculas Individuales, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias e Ingeniería, Universidad Peruana Cayetano Heredia, Lima 15102, Peru.
Open BioLab Brussels, Erasmushogeschool Brussel, Anderlecht, Brussels 1070, Belgium.
Synth Biol (Oxf). 2025 Feb 12;10(1):ysaf002. doi: 10.1093/synbio/ysaf002. eCollection 2025.
Many studies characterize transcription factors and other regulatory elements to control gene expression in recombinant systems. However, most lack a formal approach to analyse the inherent and context-specific variations of these regulatory components. This study addresses this gap by establishing a formal framework from which convenient methods are inferred to characterize regulatory circuits. We modelled the bacterial cell as a collection of proteome fractions. Deriving the time-dependent proteome fraction, we obtained a general theorem that describes its change as a function of its expression fraction, a specific portion of the total biosynthesis flux of the cell. Formal deduction reveals that when the proteome fraction reaches a maximum, it becomes equivalent to its expression fraction. This equation enables the reliable measurement of the expression fraction through direct protein quantification. In addition, the experimental data demonstrate a linear correlation between protein production rate and specific growth rate over a significant time period. This suggests a constant expression fraction within this window. For an Isopropyl β- d-1-thiogalactopyranoside (IPTG) biosensor, in five cellular contexts, expression fractions determined by the maximum method and the slope method produced strikingly similar dose-response parameters when independently fit to a Hill function. Furthermore, by analysing two more biosensors, for mercury and cumate detection, we demonstrate that the slope method can be applied effectively to various systems. Therefore, the concepts presented here provide convenient methods for obtaining dose-response parameters, clearly defining the time interval of their validity and offering a framework for interpreting typical biosensor outputs in terms of bacterial physiology. Graphical Abstract Nutrients, transformed by the action of the Nutrient Fixators (purple arrow), are used at a rate of ρ for Protein biosynthesis. The total rate ρ is multiplied by expression fractions f, f, f, and f to obtain the biosynthesis rate (black arrows) of each proteome fraction Φ, Φ, Φ, Φ, respectively. In a graph of Growth rate versus Proteome Fraction Production Rate, a linear function (green lines) can be observed, and its slope is equal to the expression fraction at each condition.
许多研究对转录因子和其他调控元件进行表征，以控制重组系统中的基因表达。然而，大多数研究缺乏一种正式的方法来分析这些调控元件的内在和特定背景下的变异。本研究通过建立一个正式框架来填补这一空白，从中推导出方便的方法来表征调控回路。我们将细菌细胞建模为蛋白质组组分的集合。通过推导随时间变化的蛋白质组组分，我们得到了一个通用定理，该定理将其变化描述为其表达分数的函数，表达分数是细胞总生物合成通量的一个特定部分。形式推导表明，当蛋白质组组分达到最大值时，它就等同于其表达分数。这个方程使得通过直接蛋白质定量来可靠地测量表达分数成为可能。此外，实验数据表明，在相当长的一段时间内，蛋白质产生速率与比生长速率之间存在线性相关性。这表明在这个窗口内表达分数是恒定的。对于异丙基-β-D-1-硫代半乳糖苷（IPTG）生物传感器，在五种细胞背景下，通过最大值法和斜率法确定的表达分数在独立拟合到希尔函数时产生了惊人相似的剂量响应参数。此外，通过分析另外两种用于汞和香豆酸盐检测的生物传感器，我们证明斜率法可以有效地应用于各种系统。因此，这里提出的概念提供了获得剂量响应参数的方便方法，明确界定了其有效性的时间间隔，并提供了一个根据细菌生理学解释典型生物传感器输出的框架。图形摘要 营养物质在营养固定剂（紫色箭头）的作用下发生转化，以速率ρ用于蛋白质生物合成。总速率ρ分别乘以表达分数f、f、f和f，以获得每个蛋白质组组分Φ、Φ、Φ、Φ的生物合成速率（黑色箭头）。在生长速率与蛋白质组组分产生速率的图中，可以观察到一个线性函数（绿色线条），其斜率等于每种条件下的表达分数。
