Engineered Biosensors from Dimeric Ligand-Binding Domains.
Engineered Biosensors from Dimeric Ligand-Binding Domains.
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DOI：
10.1021/acssynbio.8b00242
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发表时间：
2018-10-19
影响因子：
4.7
通讯作者：
Fields S
中科院分区：
文献类型：
--
作者：
Jester BW;Tinberg CE;Rich MS;Baker D;Fields S
生物传感器是许多合成生物学和代谢工程应用的重要组成部分。在这里，我们报道了第二代基于不稳定的二聚体配体结合域的啤酒酵母地高辛和黄体酮生物传感器，这些传感器经过配体诱导的稳定。该生物传感器包括一个与dna结合域融合的配体结合域单体和另一个与转录激活域融合的配体结合域单体，激活报告基因表达以响应类固醇结合和受体二聚化。引入不稳定突变到二聚体界面增加了一个数量级的生物传感器动态范围。通过计算重新设计二聚体界面和功能选择来创建具有进一步提高动态范围的异二聚体对。由地高辛和黄体酮配体结合域构建的异二聚体生物传感器作为合成的“and”门，对两种配体组合的响应比单独使用任何一种都强20倍。我们还发现了增加生物传感器对化学相似配体的敏感性或选择性的突变。这些二聚化生物传感器为逻辑门和其他应用的构建提供了额外的灵活性。
Biosensors are important components of many synthetic biology and metabolic engineering applications. Here, we report a second generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization. The biosensors, comprising one ligand-binding domain monomer fused to a DNA-binding domain and another fused to a transcriptional activation domain, activate reporter gene expression in response to steroid binding and receptor dimerization. The introduction of a destabilizing mutation to the dimer interface increased biosensor dynamic range by an order of magnitude. Computational redesign of the dimer interface and functional selections were used to create heterodimeric pairs with further improved dynamic range. A heterodimeric biosensor built from the digoxigenin and progesterone ligand- binding domains functioned as a synthetic “AND”-gate, with 20-fold stronger response to the two ligands in combination than to either one alone. We also identified mutations that increase the sensitivity or selectivity of the biosensors to chemically similar ligands. These dimerizing biosensors provide additional flexibility for the construction of logic gates and other applications.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
3.7
作者：
Chaudhury S;Berrondo M;Weitzner BD;Muthu P;Bergman H;Gray JJ
通讯作者：
Gray JJ
影响因子：
7.7
作者：
Feng, Justin;Jester, Benjamin W.;Baker, David
通讯作者：
Baker, David
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.