Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks.
Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks.
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DOI：
10.1016/j.cels.2017.06.001
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发表时间：
2017-07
期刊：
影响因子：
9.3
通讯作者：
Enoch Yeung;Aaron J. Dy;Kyle B. Martin;Andrew H. Ng;Domitilla Del Vecchio;J. Beck;J. Collins;R. Murray
中科院分区：
文献类型：
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作者：
Enoch Yeung;Aaron J. Dy;Kyle B. Martin;Andrew H. Ng;Domitilla Del Vecchio;J. Beck;J. Collins;R. Murray
合成基因表达对基因内组成环境（启动子结构、启动子与编码序列之间的间隔区域以及核糖体结合位点）高度敏感。然而，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。我们比较了诱导基因在趋同、发散或串联方向上的表达。与发散型或串联型基因相比，聚合型基因的表达量、超灵敏度和动态范围可提高400%。取向影响基因的表达，无论是诱导一个基因还是诱导两个基因。我们假设，由超卷曲介导的发散型和串联型基因的转录干扰可以解释表达差异，并通过建模和体外超卷曲放松实验验证了这一假设。用gyrase处理消除了基因间环境效应，使表达水平在30%以内。我们利用收敛基因重构拨动开关，利用超卷绕效应提高阈值检测和开关稳定性。
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 andin vitrosupercoiling 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.