Delvigne Frank, Henrion Lucas, Vandenbroucke Vincent, Martinez Juan Andres
Terra Research and Teaching Centre, Microbial Processes and Interactions (MiPI), Gembloux Agro-Bio Tech, University of Liège, Gembloux, Belgium.
Methods Mol Biol. 2023;2617:103-120. doi: 10.1007/978-1-0716-2930-7_7.
Different expression vectors are available for the effective production of recombinant proteins by bacterial populations. However, the productivity of such systems is limited by the inherent noise of the gene circuits used for the synthesis of recombinant products. An extreme case of cell-to-cell heterogeneity that has been previously reported for the ara- and lac-based expression systems in E. coli is the all-or-none response. According to this mode of response, two subpopulations of cells are generated, i.e., a "low-" subpopulation exhibiting a shallow expression level and a "high-" subpopulation exhibiting a high-expression level. The "low-" subpopulation can be considered as a cluster of non-producing cells contributing to the loss of productivity. Here we describe the setup, design, and operation of a continuous culture where inducer addition is operated based on microbial population dynamics. The determination of population dynamics is done based on an automated flow cytometry (FC) procedure previously denoted as segregostat. We illustrate how this setup can be used to control the activation of an ara-based expression system and avoid phenotypic diversification leading to an all-or-none response. Upon the determination of the natural frequency of the gene circuit used as an expression system, our current protocol can be set up without the requirement of a feedback controller.
有不同的表达载体可用于细菌群体有效生产重组蛋白。然而，此类系统的生产力受到用于合成重组产物的基因回路固有噪声的限制。先前报道的大肠杆菌中基于阿拉伯糖（ara）和乳糖（lac）的表达系统细胞间异质性的一个极端情况是全或无反应。根据这种反应模式，会产生两个细胞亚群，即表达水平较低的“低”亚群和表达水平较高的“高”亚群。“低”亚群可被视为导致生产力损失的不产生蛋白的细胞簇。在此，我们描述了一种连续培养的设置、设计和操作，其中诱导剂的添加基于微生物群体动态进行。群体动态的测定基于先前称为分选恒化器的自动流式细胞术（FC）程序。我们说明了如何使用这种设置来控制基于ara的表达系统的激活，并避免导致全或无反应的表型多样化。在确定用作表达系统的基因回路的固有频率后，我们当前的方案无需反馈控制器即可设置。
