School of Life Science, Northeast Forestry University, Harbin 150040, China.
NEFU-China iGEM Team, Northeast Forestry University, Harbin 150040, China.
Int J Mol Sci. 2024 Jan 26;25(3):1533. doi: 10.3390/ijms25031533.
Biosensors based on allosteric transcription factors have been widely used in synthetic biology. In this study, we utilized the ADP1 transcription factor PobR to develop a biosensor activating the P promoter when bound to its natural ligand, 4-hydroxybenzoic acid (4HB). To screen for PobR mutants responsive to 4-hydroxyphenylpyruvate(HPP), we developed a dual selection system in . The positive selection of this system was used to enrich PobR mutants that identified the required ligands. The following negative selection eliminated or weakened PobR mutants that still responded to 4HB. Directed evolution of the PobR library resulted in a variant where PobR was 5.1 times more reactive to 4-hydroxyphenylpyruvate than PobR. Overall, we developed an efficient dual selection system for directed evolution of biosensors.
基于别构转录因子的生物传感器已广泛应用于合成生物学。在这项研究中，我们利用 ADP1 转录因子 PobR 开发了一种生物传感器，当与天然配体 4-羟基苯甲酸（4HB）结合时，该生物传感器可激活 P 启动子。为了筛选与 4-羟基苯丙酮酸（HPP）结合的 PobR 突变体，我们在 中开发了一个双重选择系统。该系统的正选择用于富集鉴定必需配体的 PobR 突变体。接下来的负选择则消除或减弱了仍能对 4HB 做出反应的 PobR 突变体。对 PobR 文库的定向进化导致 PobR 对 4-羟基苯丙酮酸的反应性提高了 5.1 倍。总的来说，我们开发了一种用于生物传感器定向进化的高效双重选择系统。
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