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Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii

Year 2017, Volume: 20 Issue: 2, 89 - 94, 04.01.2017

Öznur Can , Hülya Kuduğ , Köksal Pabuçcu İsa Gökçe

Abstract

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

References

  • Almaraz-Delgado AL, Flores-Uribe J, Pérez-España VH, Salgado-Manjarrez E, Badillo-Corona JA 2014. Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii. AMB Express, 4: 57.
  • Balamurugan V, Reddy GR, Suryanarayana VVS 2007. Pichia pastoris: A notable heterologous expression system for the production of foreign proteins Vaccines. Indıan J Biotechnol, 6: 175-186.
  • Bertalan I, Munder MC, Weiß C, Kopf J, Fischer D, Johanningmeier U 2015. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii. J Biotechnol, 195 : 60 - 66.
  • Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein T, Shark KB, Sanford JC 1988. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science, 240: 1534 - 1538.
  • Ferrante P, Catalanotti C, Bonente G, Giuliano G 2008. An optimized, chemically regulated gene expression system for Chlamydomonas. PLos ONE, 3: e3200.
  • Franklin SE, Mayfield SP 2004. Prospects for molecular farming in the green algae Chlamydomonas reinhardtii. Curr Opın Plant Biol, 7: 159 - 65.
  • Fuhrmann M, Oertel W, Hegeman P 1999. A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J, 19: 353 - 361.
  • Gong Y, Hu H, Gao Y, Xu X, Gao H 2011. Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects. J Ind Microbial Biotechnology, 38: 1879-1890.
  • Griesbeck C, Kobl I, Heitzer M 2006. Chlamydomonas reinhardtii: a Protein Expression System for Pharmaceutical and Biotechnological Proteins. Mol Biotechnol, 34: 213 - 223.
  • Harris, E.H. 2009. The Chlamydomonas Sourcebook, 2nd ed.Oxford, UK, Academic Press.
  • Jeong B, Wu-Scharf D, Zhang C, Cerutti H 2002. Supressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements. Proc Natl Acad Sci USA, 99: 1076 - 1081.
  • Kindle KL, Schnell RA, Fernandez E, Lefebre PA 1989. Stable Nuclear Transformation of Chlamydomonas Using the Chlamydomonas Gene for Nitrate Reductase. J Cell Biol, 109: 2589 - 2601.
  • Kumar A, Falcao VR, Sayre RT 2013. Evaluating nuclear transgene expression systems in Chlamydomonas reinhardtii. Algal Res, 2: 321–332.
  • Laemmli U.K 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680 - 685.
  • Lauersen KJ, Berger H, Mussgnug JH, Kruse O 2013. Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardti. J Biotechnol, 167: 101 - 110.
  • Mayfield SP, Manuell AL, Chen S, Wu J, Tran M, Siefker D, Muto M, Marin-Navarro J 2007. Chlamydomonas reinhardtii chloroplasts as protein factories. Curr Opin in Biotechnol, 18: 1 - 8.
  • Neupert J, Shao N, Lu Y, Bock R 2012. Genetic Transformation of the Model Green Alga. Methods Mol Biol, 847: 35-47.
  • Potvin G, Zhang Z 2010. Strategies for high-level recombinant protein expression in transgenic microalgae. Biotechnol Adv, 28: 910-918.
  • Randolph-Anderson BL, Boynton JE, Gillham NW, Harris EH, Johnson AM, Dorthu MP, Matagne RF 1993. Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation. Mol Gen Genet, 236:235-244.
  • Rasala BA, Muto M, Lee P A, Jager M, Cardoso RMF, Behnke CA, Kirk P, Hokanson CA, Crea R, Mendez M, Mayfield SP 2010. Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii, Plant Biotechnol J, DOI: 10.1111/j.1467-7652.2010.00503.x.
  • Rasala B, Mayfield SP 2011. The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics. Bioeng Bugs, 2: 50-54.
  • Rasala BA, Mayfield SP 2015. Photosynthetic biomanufacturing ingreen algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynth Res, 123: 227–239.
  • Rochaix JD 1995. Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet, 29: 209-230.
  • Rosales-Mendoza S, Paz-Maldonado LMT, Soria-Guerra RE 2012. Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins:current status and perspectives. Plant Cell Rep, 31: 479-494.
  • Rosenberg JN, Oyler GA, Wilkinson L, Betenbaugh MJ 2008. A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. Curr Opin in Biotechnol, 19: 430-436.
  • Scaife MA, Nguyen GTDT, Rico J, Lambert D, Helliwell KE, Smith AG, Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. The Plant Journal, 82(3):532-546.
  • Schroda M, Beck CF, Vallon O 2002. Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. PlantJ, 31: 445-455.
  • Shimogawara K, Fujiwara S, Grossman A, Usuda H 1997. High-Efficiency Transformation of Chlamydomonas reinhardtii by Electroporation. Genetics, 148: 1821-1828.
  • Specht EA, Mayfield SP 2014. Algae-based oral recombinant vaccines. Front. Microbiol, 5:60.
  • Walker TL, Collet C, Purton S 2005. Algal transgenics in the genomic era. J. Phycol, 41: 1077-1093.

Year 2017, Volume: 20 Issue: 2, 89 - 94, 04.01.2017

Öznur Can , Hülya Kuduğ , Köksal Pabuçcu İsa Gökçe

Abstract

References

  • Almaraz-Delgado AL, Flores-Uribe J, Pérez-España VH, Salgado-Manjarrez E, Badillo-Corona JA 2014. Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii. AMB Express, 4: 57.
  • Balamurugan V, Reddy GR, Suryanarayana VVS 2007. Pichia pastoris: A notable heterologous expression system for the production of foreign proteins Vaccines. Indıan J Biotechnol, 6: 175-186.
  • Bertalan I, Munder MC, Weiß C, Kopf J, Fischer D, Johanningmeier U 2015. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii. J Biotechnol, 195 : 60 - 66.
  • Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein T, Shark KB, Sanford JC 1988. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science, 240: 1534 - 1538.
  • Ferrante P, Catalanotti C, Bonente G, Giuliano G 2008. An optimized, chemically regulated gene expression system for Chlamydomonas. PLos ONE, 3: e3200.
  • Franklin SE, Mayfield SP 2004. Prospects for molecular farming in the green algae Chlamydomonas reinhardtii. Curr Opın Plant Biol, 7: 159 - 65.
  • Fuhrmann M, Oertel W, Hegeman P 1999. A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J, 19: 353 - 361.
  • Gong Y, Hu H, Gao Y, Xu X, Gao H 2011. Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects. J Ind Microbial Biotechnology, 38: 1879-1890.
  • Griesbeck C, Kobl I, Heitzer M 2006. Chlamydomonas reinhardtii: a Protein Expression System for Pharmaceutical and Biotechnological Proteins. Mol Biotechnol, 34: 213 - 223.
  • Harris, E.H. 2009. The Chlamydomonas Sourcebook, 2nd ed.Oxford, UK, Academic Press.
  • Jeong B, Wu-Scharf D, Zhang C, Cerutti H 2002. Supressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements. Proc Natl Acad Sci USA, 99: 1076 - 1081.
  • Kindle KL, Schnell RA, Fernandez E, Lefebre PA 1989. Stable Nuclear Transformation of Chlamydomonas Using the Chlamydomonas Gene for Nitrate Reductase. J Cell Biol, 109: 2589 - 2601.
  • Kumar A, Falcao VR, Sayre RT 2013. Evaluating nuclear transgene expression systems in Chlamydomonas reinhardtii. Algal Res, 2: 321–332.
  • Laemmli U.K 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680 - 685.
  • Lauersen KJ, Berger H, Mussgnug JH, Kruse O 2013. Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardti. J Biotechnol, 167: 101 - 110.
  • Mayfield SP, Manuell AL, Chen S, Wu J, Tran M, Siefker D, Muto M, Marin-Navarro J 2007. Chlamydomonas reinhardtii chloroplasts as protein factories. Curr Opin in Biotechnol, 18: 1 - 8.
  • Neupert J, Shao N, Lu Y, Bock R 2012. Genetic Transformation of the Model Green Alga. Methods Mol Biol, 847: 35-47.
  • Potvin G, Zhang Z 2010. Strategies for high-level recombinant protein expression in transgenic microalgae. Biotechnol Adv, 28: 910-918.
  • Randolph-Anderson BL, Boynton JE, Gillham NW, Harris EH, Johnson AM, Dorthu MP, Matagne RF 1993. Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation. Mol Gen Genet, 236:235-244.
  • Rasala BA, Muto M, Lee P A, Jager M, Cardoso RMF, Behnke CA, Kirk P, Hokanson CA, Crea R, Mendez M, Mayfield SP 2010. Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii, Plant Biotechnol J, DOI: 10.1111/j.1467-7652.2010.00503.x.
  • Rasala B, Mayfield SP 2011. The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics. Bioeng Bugs, 2: 50-54.
  • Rasala BA, Mayfield SP 2015. Photosynthetic biomanufacturing ingreen algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynth Res, 123: 227–239.
  • Rochaix JD 1995. Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet, 29: 209-230.
  • Rosales-Mendoza S, Paz-Maldonado LMT, Soria-Guerra RE 2012. Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins:current status and perspectives. Plant Cell Rep, 31: 479-494.
  • Rosenberg JN, Oyler GA, Wilkinson L, Betenbaugh MJ 2008. A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. Curr Opin in Biotechnol, 19: 430-436.
  • Scaife MA, Nguyen GTDT, Rico J, Lambert D, Helliwell KE, Smith AG, Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. The Plant Journal, 82(3):532-546.
  • Schroda M, Beck CF, Vallon O 2002. Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. PlantJ, 31: 445-455.
  • Shimogawara K, Fujiwara S, Grossman A, Usuda H 1997. High-Efficiency Transformation of Chlamydomonas reinhardtii by Electroporation. Genetics, 148: 1821-1828.
  • Specht EA, Mayfield SP 2014. Algae-based oral recombinant vaccines. Front. Microbiol, 5:60.
  • Walker TL, Collet C, Purton S 2005. Algal transgenics in the genomic era. J. Phycol, 41: 1077-1093.
There are 30 citations in total.

Details

Journal Section BİYOLOJİ (Biology)
Authors

Öznur Can

Hülya Kuduğ

Köksal Pabuçcu This is me

İsa Gökçe

Publication Date January 4, 2017
Published in Issue Year 2017 Volume: 20 Issue: 2

Cite

APA Can, Ö., Kuduğ, H., Pabuçcu, K., Gökçe, İ. (2017). Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii. KSÜ Doğa Bilimleri Dergisi, 20(2), 89-94.
AMA Can Ö, Kuduğ H, Pabuçcu K, Gökçe İ. Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii. KSÜ Doğa Bilimleri Dergisi. January 2017;20(2):89-94.
Chicago Can, Öznur, Hülya Kuduğ, Köksal Pabuçcu, and İsa Gökçe. “Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas Reinhardtii”. KSÜ Doğa Bilimleri Dergisi 20, no. 2 (January 2017): 89-94.
EndNote Can Ö, Kuduğ H, Pabuçcu K, Gökçe İ (January 1, 2017) Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii. KSÜ Doğa Bilimleri Dergisi 20 2 89–94.
IEEE Ö. Can, H. Kuduğ, K. Pabuçcu, and İ. Gökçe, “Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii”, KSÜ Doğa Bilimleri Dergisi, vol. 20, no. 2, pp. 89–94, 2017.
ISNAD Can, Öznur et al. “Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas Reinhardtii”. KSÜ Doğa Bilimleri Dergisi 20/2 (January 2017), 89-94.
JAMA Can Ö, Kuduğ H, Pabuçcu K, Gökçe İ. Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii. KSÜ Doğa Bilimleri Dergisi. 2017;20:89–94.
MLA Can, Öznur et al. “Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas Reinhardtii”. KSÜ Doğa Bilimleri Dergisi, vol. 20, no. 2, 2017, pp. 89-94.
Vancouver Can Ö, Kuduğ H, Pabuçcu K, Gökçe İ. Electroporation-Mediated GFP Gene Transfer into Model Organism Chlamydomonas reinhardtii. KSÜ Doğa Bilimleri Dergisi. 2017;20(2):89-94.
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