Department of Bioengineering, University of California, Berkeley, USA.
J Biol Eng. 2011 Jul 25;5:10. doi: 10.1186/1754-1611-5-10.
Engineers frequently vary design parameters to optimize the behaviour of a system. However, synthetic biologists lack the tools to rapidly explore a critical design parameter, gene expression level, and have no means of systematically varying the dosage of an entire genetic circuit. As a step toward overcoming this shortfall, we have developed a technology that enables the same plasmid to be maintained at different copy numbers in a set of closely related cells. This provides a rapid method for exploring gene or cassette dosage effects.
We engineered two sets of strains to constitutively provide a trans-acting replication factor, either Pi of the R6K plasmid or RepA of the ColE2 plasmid, at different doses. Each DIAL (different allele) strain supports the replication of a corresponding plasmid at a constant level between 1 and 250 copies per cell. The plasmids exhibit cell-to-cell variability comparable to other popular replicons, but with improved stability. Since the origins are orthogonal, both replication factors can be incorporated into the same cell. We demonstrate the utility of these strains by rapidly assessing the optimal expression level of a model biosynthetic pathway for violecein.
The DIAL strains can rapidly optimize single gene expression levels, help balance expression of functionally coupled genetic elements, improve investigation of gene and circuit dosage effects, and enable faster development of metabolic pathways.
工程师经常改变设计参数以优化系统的行为。然而，合成生物学家缺乏快速探索关键设计参数（基因表达水平）的工具，也没有系统改变整个遗传回路剂量的方法。为了克服这一不足，我们开发了一种技术，使同一质粒能够在一组密切相关的细胞中保持不同的拷贝数。这为探索基因或盒剂量效应提供了一种快速方法。
我们设计了两组菌株，以组成型方式提供不同剂量的反式作用复制因子，要么是 R6K 质粒的 Pi，要么是 ColE2 质粒的 RepA。每个 DIAL（不同等位基因）菌株在每个细胞 1 到 250 个拷贝之间以恒定水平支持相应质粒的复制。这些质粒表现出与其他流行的复制子相当的细胞间可变性，但稳定性有所提高。由于原点是正交的，因此两种复制因子都可以整合到同一个细胞中。我们通过快速评估模型生物合成途径的最佳表达水平来证明这些菌株的实用性，该途径用于合成紫罗酮。
DIAL 菌株可以快速优化单个基因的表达水平，有助于平衡功能偶联遗传元件的表达，改善基因和电路剂量效应的研究，并加快代谢途径的开发。
