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高效转化和基因组库在酵母酵母中的表达
Mira Loock1, Luiza Berenguer Antunes1, Rhiannon T Heslop1,2
1Institute of Parasitology, McGill University, Ste Anne de Bellevue, QC H9X 3V9, Canada.
Methods and protocols
|September 22, 2023
概括
这项研究优化了酵母电对表达基因组库的优化,实现了100倍更高的效率. 改进的协议需要更少的DNA,确保用于选应用程序的库多样性.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 酵母遗传学 酵母遗传学
背景情况:
- 麦芽是一种表达基因组范围内的查图书馆的关键系统.
- 高效的酵母转化和受控的表达对于大型图书馆至关重要.
- 现有的电穿孔协议需要大量的线性化等离子体DNA,并产生较低的转换效率 (大约1. 10^6 cfu/μg) 的情况.
研究的目的:
- 优化酵母电,以实现全基因组库的高效表达.
- 开发一种能够显著提高转换效率 (高达10^8 cfu/μg) 的协议.
- 以减少基因组图书馆输入来实现强大的图书馆选.
主要方法:
- 优化了酵母细胞的电解协议.
- 使用酸和dithiothreitol调节剂制备电竞的酵母.
- 用载体DNA进行电解,然后进行纳米孔测序以验证.
主要成果:
- 实现了高达10^8 cfu/μg等离子体DNA的转换效率,这是100倍的改进.
- 协议需要最小的基因组图书馆DNA (每次反应0.1μg) 进行10-100×图书馆覆盖.
- 纳米孔测序证实了图书馆大小和多样性的保存.
- 表达式分析验证了库的功能和协议的有效性.
结论:
- 优化的协议显著提高了酵母转化效率,用于基因组图书馆表达.
- 它允许使用减少数量的起始基因组DNA进行全面的图书馆选.
- 这种方法为酵母下游选应用提供了强大而高效的方法.
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