Autolysin Production from Chlamydomonas reinhardtii.

Justin Findinier
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Abstract

Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.

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莱茵衣藻产自溶素。
莱茵衣藻是多种过程的模式生物,从光合作用到纤毛生物发生,是了解更多生物燃料生产的良好基础。这是由于可用的分子工具的宽度,最近随着模块化克隆系统的发展而扩大,但最重要的是,随着CRISPR/Cas9编辑现在成为可能。该技术已被证明在没有细胞壁的情况下更有效,通过使用特定的突变体或使用交配特异性金属蛋白酶自溶素(也称为配子化素)消化衣藻细胞壁。从衣藻细胞生产自溶素已经使用和共享了多种方案;然而,它们提供了非常不一致的结果,这阻碍了常规执行CRISPR诱变的能力。在这里,我们提出了一种简单的自溶素生产方案,需要将细胞从培养皿转移到密集的液体悬浮液中,在混合配子之前通过过夜孵育进行配子形成,并在2小时后收获酶。该方案已被证明是高效的自溶素生产,而无需在任何步骤精确控制细胞密度。只需最少的人工,它将提供一种简单、现成的方法来生产一种对目标突变体产生至关重要的酶。
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