An Iterative, Synthetic Approach To Engineer a High-Performance PhoB-Specific Reporter
An Iterative, Synthetic Approach To Engineer a High-Performance PhoB-Specific Reporter
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设计高性能 PhoB 特异性报告基因的迭代综合方法
DOI：
10.1128/aem.00603-18
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发表时间：
2018
影响因子：
4.4
通讯作者：
Parales, Rebecca E.
中科院分区：
文献类型：
--
作者：
Stoudenmire, Julie L.;Essock-Burns, Tara;Weathers, Erena N.;Solaimanpour, Sina;Mrázek, Jan;Stabb, Eric V.;Parales, Rebecca E.
转录报告基因是分析目的基因转录或特定转录调节因子活性的常用工具。不幸的是，后一种应用具有天然启动子没有进化为特定调节剂活性的最佳读数的缺点。我们试图合成一个优化的转录报告评估PhoB的活性，旨在最大的“开”的表达时，PhoB是活跃的，最小的背景下的“关”状态，没有其他监管机构的控制元件。我们为具有适当间隔的PhoB结合位点的启动子元件设计了特定序列，并且在我们没有预测序列特定效应的另外19个插入核苷酸位置处，碱基是随机的。在费氏弧菌中筛选了83个这样的构建体，使我们能够识别与高水平“开”或低水平“关”表达显著相关的特定随机位置处的碱基。第二轮启动子设计合理地限制了13个额外的位置，导致具有高水平PhoB依赖性表达的报告子，基本上没有背景，也没有其他已知的调控元件。作为表达的报告基因，我们使用稳定的和不稳定的绿色荧光蛋白（GFP）变体，后者在费氏弧菌中的半衰期为81分钟。在培养物中，当磷酸盐耗尽至低于10 μM的浓度时，PhoB诱导报告基因。在共生殖民的主机鱿鱼，Euprymna candiopes，记者表示在不同的光器官微环境异质磷酸盐的可用性。最后，在变形菌门的其他成员中测试该构建体证明了其更广泛的用途。结果说明了如何有限的能力来预测合成的启动子报告的性能可以克服通过迭代筛选和reengineering.IMPORTANCETranscriptional记者可以评估时，一个特定的调节器是活跃的强大的工具，但是，本地启动子可能不是理想的为此目的。最佳的报告应该是特定的监管机构正在审查，并应最大限度地提高“开”和“关”状态之间的差异，然而，这些属性是不同的选择性压力驱动的自然启动子的演变。合成启动子提供了一个有前途的替代方案，但我们的理解往往不能完全预测启动子设计，大量的替代序列的可能性可能是棘手的。在具有超过340亿个序列变体的合成启动子区域中，我们通过仅筛选83个候选物来鉴定与有利性能相关的碱基，从而使我们能够合理地限制我们的设计。因此，我们产生了一个优化的报告，诱导的PhoB和使用它来探索低磷酸盐反应的V. fischeri。这种启动子设计策略将促进其他调控特异性报告基因的工程化。
Transcriptional reporters are common tools for analyzing either the transcription of a gene of interest or the activity of a specific transcriptional regulator. Unfortunately, the latter application has the shortcoming that native promoters did not evolve as optimal readouts for the activity of a particular regulator. We sought to synthesize an optimized transcriptional reporter for assessing PhoB activity, aiming for maximal “on” expression when PhoB is active, minimal background in the “off” state, and no control elements for other regulators. We designed specific sequences for promoter elements with appropriately spaced PhoB-binding sites, and at 19 additional intervening nucleotide positions for which we did not predict sequence-specific effects, the bases were randomized. Eighty-three such constructs were screened in Vibrio fischeri, enabling us to identify bases at particular randomized positions that significantly correlated with high-level “on” or low-level “off” expression. A second round of promoter design rationally constrained 13 additional positions, leading to a reporter with high-level PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we used both stable and destabilized variants of green fluorescent protein (GFP), the latter of which has a half-life of 81 min in V. fischeri. In culture, PhoB induced the reporter when phosphate was depleted to a concentration below 10 μM. During symbiotic colonization of its host squid, Euprymna scolopes, the reporter indicated heterogeneous phosphate availability in different light-organ microenvironments. Finally, testing this construct in other members of the Proteobacteria demonstrated its broader utility. The results illustrate how a limited ability to predict synthetic promoter-reporter performance can be overcome through iterative screening and reengineering.IMPORTANCETranscriptional reporters can be powerful tools for assessing when a particular regulator is active; however, native promoters may not be ideal for this purpose. Optimal reporters should be specific to the regulator being examined and should maximize the difference between the “on” and “off” states; however, these properties are distinct from the selective pressures driving the evolution of natural promoters. Synthetic promoters offer a promising alternative, but our understanding often does not enable fully predictive promoter design, and the large number of alternative sequence possibilities can be intractable. In a synthetic promoter region with over 34 billion sequence variants, we identified bases correlated with favorable performance by screening only 83 candidates, allowing us to rationally constrain our design. We thereby generated an optimized reporter that is induced by PhoB and used it to explore the low-phosphate response of V. fischeri. This promoter design strategy will facilitate the engineering of other regulator-specific reporters.
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