Document Type : Article
Iranian Research Organization for Science and Technology
Iranian research organization for science and technology (IROST)
Iranian research organization for science and technology
Background: Chlamydomonas reinhardtii is a novel recombinant eukaryotic expression system with many advantages including fast growth rate, rapid scalability, absence of human pathogens and the ability to fold and assemble complex proteins accurately, however, obstacle relatively low expression level necessitates optimizing foreign gene expression in this system. The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a substantial reporter molecule for monitoring gene expression and protein localization.The fluorescence of GFP requires only UV or blue light, and, therefore, the in vivo observation of GFP expression is easy with no need for complex and costly apparatus.
Methods: In this study the codon optimized GFP reporter gene was cloned into the pChlamy3 vector using Kpn1 and Not1 restriction sites. The recombinant pChlamy3-GFP expression vector was transformed into E. coli Top10 cells. The presence of the expression cassette was verified by double digestion. Then the recombinant vector was transformed into the nucleus of C. reinhardtii for expression. Afterwards, transformed cells were analyzed by fluorescence spectroscopy using a microplate reader.
Results: The results of fluorescence spectroscopy revealed a 28-fold more fluorescence compared to wild type in one of the samples, and this is much more than the reported results by previous studies.
Conclusions: It is suggested that the expression system optimized by this study can potentially be used for the production of important therapeutic proteins and other heterologous proteins in C. reinhardtii.