Mauri Marco, Vecchione Stefano, Fritz Georg
LOEWE Center for Synthetic Microbiology and Department of Physics , Philipps-Universität Marburg , 35032 Marburg , Germany.
ACS Synth Biol. 2019 Jun 21;8(6):1361-1370. doi: 10.1021/acssynbio.9b00032. Epub 2019 May 29.
Luciferase reporters have become standard genetic tools to monitor gene expression in real time and in high-throughput using microplate readers. Compared to reporter gene assays based on fluorescence proteins, luciferase reporters have a superior signal-to-noise ratio, since they do not suffer from the high autofluorescence background of the bacterial cell. However, at the same time luciferase reporters have the drawback of constant light emission, which leads to undesired cross-talk between neighboring wells on a microplate. To overcome this limitation, we developed a computational method to correct for luminescence bleed-through and to estimate the "true" luminescence activity for each well of a microplate. As the sole input our algorithm uses the signals measured from a calibration plate, in which the light emitted from a single luminescent well serves as an estimate for the "light-spread function". We show that this light-spread function can be used to deconvolve any other measurement obtained under the same technical conditions. Our analysis demonstrates that the correction preserves low-level signals close to the background and shows that it is universally applicable to different kinds of microplate readers and plate types.
荧光素酶报告基因已成为使用微孔板读数器实时、高通量监测基因表达的标准遗传工具。与基于荧光蛋白的报告基因检测相比，荧光素酶报告基因具有更高的信噪比，因为它们不受细菌细胞高自发荧光背景的影响。然而，与此同时，荧光素酶报告基因存在持续发光的缺点，这会导致微孔板上相邻孔之间产生不必要的串扰。为克服这一限制，我们开发了一种计算方法，用于校正发光渗漏，并估计微孔板每个孔的“真实”发光活性。作为唯一输入，我们的算法使用从校准板测量的信号，其中单个发光孔发出的光用作“光扩散函数”的估计值。我们表明，这种光扩散函数可用于对在相同技术条件下获得的任何其他测量值进行去卷积。我们的分析表明，校正保留了接近背景的低水平信号，并表明它普遍适用于不同类型的微孔板读数器和板型。
