David Lidor, Shpigel Etai, Levin Itay, Moshe Shaked, Zimmerman Lior, Dadon-Simanowitz Shilat, Shemer Benjamin, Levkovich Shon A, Larush Liraz, Magdassi Shlomo, Belkin Shimshon
Enzymit Ltd. 3 Pinhas Sapir St., Ness Ziona 7403626, Israel.
Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
Comput Struct Biotechnol J. 2023 Aug 22;21:4252-4260. doi: 10.1016/j.csbj.2023.08.017. eCollection 2023.
We present a methodology for a high-throughput screening (HTS) of transcription factor libraries, based on bacterial cells and GFP fluorescence. The method is demonstrated on the LysR-type transcriptional regulator YhaJ, a key element in 2,4-dinitrotuluene (DNT) detection by bacterial explosives' sensor strains. Enhancing the performance characteristics of the YhaJ transcription factor is essential for future standoff detection of buried landmines. However, conventional directed evolution methods for modifying YhaJ are limited in scope, due to the vast sequence space and the absence of efficient screening methods to select optimal transcription factor mutants. To overcome this limitation, we have constructed a focused saturation library of ca. 6.4 × 107 variants, and have screened over 70 % of its sequence space using fluorescence-activated cell sorting (FACS). Through this screening process, we have identified YhaJ mutants exhibiting superior fluorescence responses to DNT, which were then effectively transformed into a bioluminescence-based DNT detection system. The best modified DNT reporter strain demonstrated a 7-fold lower DNT detection threshold, a 45-fold increased signal intensity, and a 40 % shorter response time compared to the parental bioreporter. The FACS-based HTS approach presented here may hold a potential for future molecular enhancement of other sensing and catalytic bioreactions.
我们提出了一种基于细菌细胞和绿色荧光蛋白（GFP）荧光的转录因子文库高通量筛选（HTS）方法。该方法在LysR型转录调节因子YhaJ上得到了验证，YhaJ是细菌爆炸物传感器菌株检测2,4-二硝基甲苯（DNT）的关键元件。增强YhaJ转录因子的性能特征对于未来远距离探测埋藏地雷至关重要。然而，由于巨大的序列空间以及缺乏有效的筛选方法来选择最佳转录因子突变体，传统的用于修饰YhaJ的定向进化方法在范围上受到限制。为了克服这一限制，我们构建了一个约6.4×10^7个变体的聚焦饱和文库，并使用荧光激活细胞分选（FACS）筛选了其70%以上的序列空间。通过这个筛选过程，我们鉴定出了对DNT表现出优异荧光响应的YhaJ突变体，然后将其有效地转化为基于生物发光的DNT检测系统。与亲本生物报告菌株相比，最佳修饰的DNT报告菌株的DNT检测阈值降低了7倍，信号强度提高了45倍，响应时间缩短了40%。本文介绍的基于FACS的HTS方法可能对未来其他传感和催化生物反应的分子增强具有潜力。
