Data Science and Analytics Group, Computational and Statistical Analytics Division, Pacific Northwest National Laboratory, Richland, WA, USA.
Department of Biological Engineering, Institute for Medical Engineering & Science, Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Cell Syst. 2017 Jul 26;5(1):11-24.e12. doi: 10.1016/j.cels.2017.06.001. Epub 2017 Jul 19.
Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability.
基因表达的合成对基因内组成环境（启动子结构、启动子和编码序列之间的间隔区以及核糖体结合位点）非常敏感。然而，关于基因间组成环境（整个基因在 DNA 上的空间排列和方向）对合成基因网络中表达水平的影响知之甚少。我们比较了以会聚、发散或串联方向排列的诱导基因的表达。与发散或串联定向基因相比，会聚基因的诱导表达可提高高达 400%的表达水平、更高的超敏性和动态范围。无论一个或两个基因是否被诱导，方向都会影响基因表达。我们假设，通过超螺旋介导的发散和串联基因中的转录干扰可以解释表达差异，并通过建模和体外超螺旋松弛实验验证这一假设。用拓扑异构酶处理消除了基因间组成环境的影响，使表达水平彼此相差 30%。我们利用超螺旋效应，以会聚基因重建了 toggle 开关，从而提高了阈值检测和开关稳定性。
