通过将分泌应激生物传感器菌株与体内分裂的GFP试验结合起来,分析Bacillus subtilis中的蛋白质分泌
Patrick Lenz1,2, Patrick J Bakkes2,3, Carolin Müller2,3
1Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich, 52425, Jülich, Germany.
Microbial cell factories
|October 7, 2023
概括
我们为Bacillus subtilis开发了一个新的系统来监测蛋白质分泌. 这种组合的GFP和生物传感器方法允许独立于活动的检测和优化重组蛋白生产,改进工业生物技术应用.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 微生物工程 微生物工程
背景情况:
- 细菌细菌是工业生物技术中的一个关键生物体,以其蛋白质分泌能力而闻名.
- 优化重组蛋白分泌通常依赖于基于活动的查,这并不适用于所有人.
- 为了全面优化,需要一种结合活动独立检测和分泌应激监测的新系统.
研究的目的:
- 开发和验证Bacillus subtilis蛋白质分泌的联合在线监测系统.
- 为了使用分割GFP测定,使分泌蛋白质的活性独立检测.
- 使用基于mCherry的生物传感器来评估和管理分泌应激.
主要方法:
- 使用CRISPR/Cas9来表达mCherry,构建一个Bacillus subtilis生物传感器菌株 (PAL5).
- 通过将目标蛋白与GFP11融合并与GFP1-10.0补充,开发一种体内分裂GFP试验.
- 使用了一个模型酶,Fusarium solani pisi cutinase (Cut),与GFP11 (Cut11) 融合,与不同的信号.
主要成果:
- 成功构建了生物传感器菌株,并证明了分裂GFP试验的有效性.
- 显示了分裂的GFP信号和分泌的丁酶的酶活性之间的强烈相关性.
- 确定了新的信号变体,可以提高分泌效率,同时最大限度地减少压力.
结论:
- 联合的分裂GFP和分泌应激生物传感器系统允许在线检测B. subtilis中的细胞外蛋白质.
- 该系统有效地识别了蛋白质分泌过程中的瓶.
- 开发的平台有助于监测各种表达策略引起的分泌应激.
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