State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, China.
Lett Appl Microbiol. 2011 Jun;52(6):634-41. doi: 10.1111/j.1472-765X.2011.03051.x. Epub 2011 Apr 21.
To develop a reliable and sensitive high-throughput approach for the screening of engineered constitutive promoters in the yeast Pichia pastoris.
The yeast-enhanced green fluorescent protein (yEGFP) was used as the reporter to monitor the promoter strength. After eliminating the interfering components (yeast extract and tryptone) with fluorescence signal from the medium, a high-throughput screening approach was established and optimized to obtain a low standard deviation of cell density (6.9%) and fluorescence (7.4%) in 48-deep-well microplates. Then, 300 clones containing GAP promoter (P(GAP)) variants were screened, exhibiting a wide range in fluorescent intensity from about 8% to 218% of that obtained with P(GAP). Six representative clones with unique promoter sequence were picked for further characterization. A good correlation between yEGFP fluorescence in microplates and shake flasks was observed. Furthermore, the high correlation between fluorescence and transcript levels confirmed that expression was transcriptionally controlled.
We developed a reliable high-throughput screening approach that can be used to select engineered constitutive promoters of varying strengths.
This approach is expected to accelerate the selection of constitutive promoters in P. pastoris and can also be applied for the screening of other constitutive expression clones.
开发一种可靠且灵敏的高通量方法，用于筛选毕赤酵母中的工程组成型启动子。
使用酵母增强型绿色荧光蛋白（yEGFP）作为报告基因来监测启动子强度。在消除了培养基中荧光信号的干扰成分（酵母提取物和胰蛋白胨）后，建立并优化了一种高通量筛选方法，以在 48 孔深孔板中获得低细胞密度（6.9%）和荧光（7.4%）标准偏差。然后，筛选了 300 个包含 GAP 启动子（P(GAP)) 变体的克隆，其荧光强度范围从 P(GAP) 的约 8%到 218%不等。选择了六个具有独特启动子序列的代表性克隆进行进一步表征。在微孔板和摇瓶中观察到 yEGFP 荧光之间存在良好的相关性。此外，荧光和转录水平之间的高度相关性证实了表达是转录控制的。
我们开发了一种可靠的高通量筛选方法，可用于选择不同强度的工程组成型启动子。
该方法有望加速毕赤酵母中组成型启动子的选择，也可用于筛选其他组成型表达克隆。
