Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii

Yıl 2017, Cilt: 20 Sayı: 2, 89 - 94, 04.01.2017

Öznur Can , Hülya Kuduğ , Köksal Pabuçcu İsa Gökçe

Öz

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 Aracılığıyla GFP Geninin Transferi

 

ÖZET : Son on yılda, mikroalgler değerli bileşiklerin doğal kaynağı ve rekombinant protein üretimi için biyoreaktör olmaları sebebiyle dikkat çekmektedirler. Güvenli ve ucuz protein üretimi için, mikroalg tabanlı biyoreaktörler yeni yeni kullanılmaktadır. Özellikle de kısa üreme süresine, hızlı büyüme oranına, çoklu genetik sisteme ve kompleks proteinlerin aktivite kazanmaları için gereken post translasyonel modifikasyon mekanizmalarına sahip tek hücreli mikroalg türü Chlamydomonas reinhardtii öne çıkmaktadır. Bu çalışmada, biyoteknolojik çalışmalarda evrensel markör olarak kullanılan GFP genini içeren konstrükt pChlamy_3-GFP plazmidi, Chlamydomonas reinhardtii CC-125 suşunun nüklear genomuna elektroporasyon yöntemi ile aktarılmıştır. Transformantların moleküler ve genetik  analizleri, GFP geninin yüksek miktarda belirgin bir ekspresyon seviyesi olmamasına rağmen nüklear genomun stabil olarak transforme edildiğini, transgenlerin, alg kromozomal DNA'sına başarılı bir şekilde entegre olduğunu göstermiştir. Kodon optimizasyonu, promotör seçimi, intron ve UTR'ler, endojen enhansır elemanları, düzenleyici mekanizmalar, proteinlerin lokalizasyonu, posttranslasyonel modifikasyonlar ve proteaz aktiviteleri, düşük ekspresyon seviyesinin altında yatan muhtemel nedenlerdir. 

Anahtar kelimeler: Chlamydomonas reinhardtii, biyoreaktör, yeşil floresan protein, elektroporasyon

Kaynakça

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