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首页> 外文期刊>Applied Microbiology >Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae
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Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

机译：双色监控克服了快速增长的微生物中单个生物发光报道分子的局限性，并揭示了酿酒酵母代谢过程中阶段依赖性蛋白质的生产力。

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Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases.
机译：萤光素酶是有用的，无创的基因调节报告基因，可以长时间连续监测。但是，由于快速变化的细胞数量和代谢状态也会影响生物发光，因此它在快速增长的微生物（如细菌和酵母菌）中存在问题，从而混淆了报告信号。在这里，我们显示出可以通过同时监测两种不同颜色的甲虫萤光素酶的生物发光来解决发芽酵母酿酒酵母中的这些问题，其中一种颜色（绿色）报告了感兴趣的基因的活性，而第二种颜色（红色）稳定地表达并用于持续标准化绿色生物发光的信号强度波动与基因调控无关。我们将这种双重荧光素酶策略与光诱导启动子系统结合使用，以测试酵母菌呼吸振荡的不同阶段是否比其他阶段更适合于异源蛋白质的生产。通过使用光脉冲来激活绿色荧光素酶的产生，同时将信号变化标准化为红色荧光素酶，我们显示出酵母代谢周期的早期还原阶段比其他阶段产生更多的荧光素酶。

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