Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii

Over the last decade, microalgae has drawn attention as a natural source of valuable compounds and as bioreactors for recombinant protein production. Microalgae-based bioreactor is newly employed for production of safe and cost effective proteins. Especially, Chlamydomonas reinhardtii , an unicellular microalga, is the most prominent species which has a short generation time, fast growth rate, multiple genetic systems, and ability to perform posttranslational modifications machinery that plays a significant role in regulating the activity of complex proteins. In this research, the nuclear genome of the Chlamydomonas reinhardtii CC-125 strain was transformed by electroporation using construct plasmid pChlamy_3-GFP containing the gene coding for Green Fluorescent Protein (GFP) which is commonly used as an universal marker in biotechnological studies. Molecular and genetic analyses conducted on transformants revealed that the nuclear genome was stably transformed and the transgenes were integrated into the algal chromosomal DNA succesfully, albeit there was no distinct expression level of GFP gene in producing large amounts of protein. Codon optimization, choice of promoters, introns and UTRs, endogenous enhancer elements, regulatory mechanisms, localization of proteins, posttranslational modifications and  protease activities are the possible underlying causes of the low expression level. Key words: Chlamydomonas reinhardtii, bioreactor, green fluorescent protein, electroporation Model Organizma Chlamydomonas reinhardtii 'ye Elektroporasyon Araciligiyla GFP Geninin Transferi OZET : Son on yilda, mikroalgler degerli bilesiklerin dogal kaynagi ve rekombinant protein uretimi icin biyoreaktor olmalari sebebiyle dikkat cekmektedirler. Guvenli ve ucuz protein uretimi icin, mikroalg tabanli biyoreaktorler yeni yeni kullanilmaktadir. Ozellikle de kisa ureme suresine, hizli buyume oranina, coklu genetik sisteme ve kompleks proteinlerin aktivite kazanmalari icin gereken post translasyonel modifikasyon mekanizmalarina sahip tek hucreli mikroalg turu Chlamydomonas reinhardtii one cikmaktadir. Bu calismada, biyoteknolojik calismalarda evrensel markor olarak kullanilan GFP genini iceren konstrukt pChlamy_3-GFP plazmidi, Chlamydomonas reinhardtii CC-125 susunun nuklear genomuna elektroporasyon yontemi ile aktarilmistir. Transformantlarin molekuler ve genetik  analizleri, GFP geninin yuksek miktarda belirgin bir ekspresyon seviyesi olmamasina ragmen nuklear genomun stabil olarak transforme edildigini, transgenlerin, alg kromozomal DNA'sina basarili bir sekilde entegre oldugunu gostermistir. Kodon optimizasyonu, promotor secimi, intron ve UTR'ler, endojen enhansir elemanlari, duzenleyici mekanizmalar, proteinlerin lokalizasyonu, posttranslasyonel modifikasyonlar ve proteaz aktiviteleri, dusuk ekspresyon seviyesinin altinda yatan muhtemel nedenlerdir.  Anahtar kelimeler : Chlamydomonas reinhardtii, biyoreaktor, yesil floresan protein, elektroporasyon