Rhea Katherine A, McDonald Nathan D, Cole Stephanie D, Noireaux Vincent, Lux Matthew W, Buckley Patricia E
US Army Combat Capabilities Development Command Chemical Biological Center, Aberdeen Proving Ground, MD, USA.
School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, USA.
Synth Biol (Oxf). 2022 Aug 2;7(1):ysac011. doi: 10.1093/synbio/ysac011. eCollection 2022.
Cell-free expression systems provide a suite of tools that are used in applications from sensing to biomanufacturing. One of these applications is genetic circuit prototyping, where the lack of cloning is required and a high degree of control over reaction components and conditions enables rapid testing of design candidates. Many studies have shown utility in the approach for characterizing genetic regulation elements, simple genetic circuit motifs, protein variants or metabolic pathways. However, variability in cell-free expression systems is a known challenge, whether between individuals, laboratories, instruments, or batches of materials. While the issue of variability has begun to be quantified and explored, little effort has been put into understanding the implications of this variability. For genetic circuit prototyping, it is unclear when and how significantly variability in reaction activity will impact qualitative assessments of genetic components, e.g. relative activity between promoters. Here, we explore this question by assessing DNA titrations of seven genetic circuits of increasing complexity using reaction conditions that ostensibly follow the same protocol but vary by person, instrument and material batch. Although the raw activities vary widely between the conditions, by normalizing within each circuit across conditions, reasonably consistent qualitative performance emerges for the simpler circuits. For the most complex case involving expression of three proteins, we observe a departure from this qualitative consistency, offering a provisional cautionary line where normal variability may disrupt reliable reuse of prototyping results. Our results also suggest that a previously described closed loop controller circuit may help to mitigate such variability, encouraging further work to design systems that are robust to variability. Graphical Abstract.
无细胞表达系统提供了一系列工具，这些工具应用于从传感到生物制造等诸多领域。其中一个应用是基因电路原型设计，在此过程中无需克隆，并且对反应组件和条件的高度控制能够快速测试候选设计。许多研究已表明该方法在表征基因调控元件、简单基因电路基序、蛋白质变体或代谢途径方面具有实用性。然而，无细胞表达系统的变异性是一个已知的挑战，无论是在个体、实验室、仪器之间，还是在材料批次之间。虽然变异性问题已开始得到量化和探索，但在理解这种变异性的影响方面投入的精力很少。对于基因电路原型设计，尚不清楚反应活性的变异性何时以及在多大程度上会对基因组件的定性评估产生影响，例如启动子之间的相对活性。在这里，我们通过使用表面上遵循相同方案但因人员、仪器和材料批次而异的反应条件，评估七个复杂度不断增加的基因电路的DNA滴定来探索这个问题。尽管不同条件下的原始活性差异很大，但通过在每个电路内对不同条件进行归一化处理，较简单的电路会出现合理一致的定性性能。对于涉及三种蛋白质表达的最复杂情况，我们观察到这种定性一致性出现了偏差，这为正常变异性可能破坏原型设计结果的可靠复用提供了一条临时的警示线。我们的结果还表明，先前描述的闭环控制器电路可能有助于减轻这种变异性，这鼓励进一步开展工作来设计对变异性具有鲁棒性的系统。图形摘要。
