TY  - JOUR
TI  - Autolysin Production from Chlamydomonas reinhardtii
AU  - Findinier, Justin 
VL  - 13
IS  - 13
PY  - 2023
DA  - 2023/07/05
SP  - e4705
C1  - Bio-protocol 2023;13:e4705
DO  - 10.21769/BioProtoc.4705
UR  - https://doi.org/10.21769/BioProtoc.4705
AB  - 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.				Graphical overview Workflow for autolysin production from Chlamydomonas reinhardtii
KW  - Enzyme
KW  - Autolysin
KW  - Cell wall
KW  - Chlamydomonas
KW  - CRISPR
KW  - Transformation
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False