发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2179 浏览次数: 11670
相关实验方案
通过在体内直接组装多个DNA片段在青蒿中异源生产青蒿素
Nur Kusaira Khairul Ikram [...] Henrik Toft Simonsen
2023年07月20日 1159 阅读
Abstract
A common method to investigate the function of genes putatively involved in carotenoid biosynthesis is the so called color complementation assay in Escherichia coli (see, e.g., Cunningham and Gantt, 2007). In this assay, the gene under investigation is expressed in E. coli strains genetically engineered to synthesize potential carotenoid substrates, followed by analysis of the pigment changes in the carotenogenic bacteria via high-performance liquid chromatography (HPLC). Two crucial steps in this method are (i) the quantitative extraction of the carotenoids out of E. coli and (ii) the reproducible and complete separation of the pigments by HPLC.
Here, we present a protocol for the extraction and analysis of carotenoids with a broad range of polarities from carotenogenic E. coli. The solvent mixture used for extraction keeps both the lipophilic carotenes and the more polar xanthophylls in solution and is compatible with the eluent gradient of the subsequent HPLC analysis. The C30-column used is particularly suitable for the separation of various cis-isomers of carotenoids, but also for separation of stereoisomers such as α- and β-carotene or lutein and zeaxanthin.
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文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Blatt, A. and Lohr, M. (2017). Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays. Bio-protocol 7(6): e2179. DOI: 10.21769/BioProtoc.2179.
分类
植物科学 > 植物新陈代谢 > 其它化合物
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