Fakultät für Physik, Ludwig-Maximilians-Universität, Geschwister-Scholl-Platz 1, D-80539 München, Germany.
Integr Biol (Camb). 2012 May;4(5):494-501. doi: 10.1039/c2ib00102k. Epub 2012 Apr 5.
Cell-free in vitro expression is increasingly important for high-throughput expression screening, high yield protein production and synthetic biology applications. Yet its potential for quantitative investigation of gene expression and regulatory circuits is limited by the availability of data on composition, kinetic rate constants and standardized computational tools for modeling. Here we report on calibration measurements and mathematical modeling of a reconstituted in vitro expression system. We measured a series of GFP expression and mRNA transcription time courses under various initial conditions and established the translation step as the bottle neck of in vitro protein synthesis. Cell-free translation was observed to expire after 3 h independent of initial template DNA concentration. We developed a minimalistic rate equation model and optimized its parameters by performing a concurrent fit to measured time courses. The model predicts the dependence of protein yield not only on template DNA concentration, but also on experimental timing and hence is a valuable tool to optimize yield strategies.
无细胞体外表达技术在高通量表达筛选、高产蛋白生产和合成生物学应用中变得越来越重要。然而，其在定量研究基因表达和调控回路方面的潜力受到组成、动力学速率常数以及用于建模的标准化计算工具的数据可用性的限制。在这里，我们报告了一个体外表达系统的校准测量和数学建模。我们在各种初始条件下测量了一系列 GFP 表达和 mRNA 转录的时程，并确定翻译步骤是体外蛋白质合成的瓶颈。无细胞翻译在独立于初始模板 DNA 浓度的情况下，在 3 小时后被观察到耗尽。我们开发了一个简约的速率方程模型，并通过对测量的时程进行并行拟合来优化其参数。该模型不仅预测了蛋白质产量对模板 DNA 浓度的依赖性，还预测了实验时间的依赖性，因此是优化产量策略的有价值工具。
