Korean Chemical Engineering Research

  • 제56권1호
  • /
  • Pages.79-84
  • /
  • 2018
  • /
  • 0304-128X(pISSN)
  • /
  • 2233-9558(eISSN)
한국화학공학회 (The Korean Institute of Chemical Engineers)
권호 표지
DOI QR코드

DOI QR Code

디지털 전기천공을 이용한 미세조류 내 단백질 전달 연구

Delivery of Protein into Microalgae by the Digital Electroporation

  • 임도진 (국립부경대학교 화학공학과)
  • Im, Do Jin (Department of Chemical Engineering, Pukyong National University)
  • 투고 : 2017.09.22
  • 심사 : 2017.10.16
  • 발행 : 2018.02.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구에서는 기 개발된 액적 접촉충전 기반의 디지털 전기천공 기술을 이용해 미세조류에 단백질을 전달하는 연구를 수행 하였다. Chlamydomonas reinhardtii 중 세포벽이 존재하는 야생종 cc-125에 적용한 결과, 살아 있는 세포의 핵 내부로 형광 단백질 GFP가 10% 이상의 비교적 높은 효율로 전달될 수 있음을 확인하였다. 또한 인가 전기장의 크기 변화에 따른 단백질 전달 효율을 살펴봄으로써 최적의 단백질 전달 효율을 위한 전기천공 전기장 조건을 도출하였다(960 V/cm). 전달 물질의 크기에 따른 영향 분석을 위해 추가로 수행한 핵산 염색 형광 염료 Yo-Pro-1의 전달 특성 분석 결과, 크기에 따른 차이가 존재함에도 최적의 전달 효율을 나타내는 인가 전기장의 세기 조건은 매우 유사한 경향을 보였다. 마지막으로 본 연구 결과의 의미 및 크리스퍼 유전자 가위 기술의 적용 등 향후 활용방안에 대해서 논의하였다.

In the present study, we performed electroporation to deliver protein into microalgae using previously developed digital electroporation system. Green fluorescence protein was successfully delivered into a live microalgae cell nucleus without cell wall removal. By investigating the effects of applied voltage on the protein delivery efficiency, optimal electroporation electric field condition was found (960 V/cm). We also investigated the delivery of Yo-Pro-1 into cell to examine the size effects of delivered materials and found that there is little size effects on the optimal condition. Finally, the implications of the present results and future work are discussed.

키워드

참고문헌

  1. Wijffels, R. H. and Barbosa, M. J., An "Outlook on Microalgal Biofuels," Science 329, 796-799(2010). https://doi.org/10.1126/science.1189003
  2. Pires, J. C. M., Alvim-Ferraz, M. C. M., Martins, F. G. and Simoes, M., "CarboN Dioxide Capture From Flue Gases Using Microalgae: Engineering Aspects and Biorefinery Concept," Renew. Sust. Energ. Rev. 16, 3043-3053 (2012). https://doi.org/10.1016/j.rser.2012.02.055
  3. Guo, S. L., Zhao, X. Q., Tang, Y., Wan, C., Alam, M. A., Ho, S. H., Bai, F. W. and Chang, J. S., "Establishment of an Efficient Genetic Transformation System in Scenedesmus Obliquus," J. Biotechnol. 163, 61-68(2013). https://doi.org/10.1016/j.jbiotec.2012.10.020
  4. Gimpel, J. A., Specht, E. A., Georgianna, D. R. and Mayfield, S. P., "Advances in Microalgae Engineering and Synthetic Biology Applications for Biofuel Production," Curr. Opin. Chem. Biol. 17, 489-495(2013). https://doi.org/10.1016/j.cbpa.2013.03.038
  5. Scranton, M. A., Ostrand, J. T., Fields, F. J. and Mayfield, S. P. "Chlamydomonas as a Model for Biofuels and Bio-products Production," Plant J. 82, 523-531(2015). https://doi.org/10.1111/tpj.12780
  6. Specht, E. A., Miyake-Stoner, S. and Mayfield S. P., "Micro-algae Come of Age as a Platform for Recombinant Protein Production," Biotech. Lett. 32, 1373-1383(2010). https://doi.org/10.1007/s10529-010-0326-5
  7. Jo, J., Shin, S., Jung, H., Min, B., Kim, S. and Kim, J., "Process Development for Production of Antioxidants from Lipid Extracted Microalgae Using Ultrasonic-assisted Extraction," Korean Chem. Eng. Res., 55(4), 542-547(2017). https://doi.org/10.9713/KCER.2017.55.4.542
  8. Shimogawara, K., Fujiwara, S., Grossman, A., Usuda, H., "High-Efficiency Transformation of Chlamydomonas Reinhardtii by Electroporation," Genetics 148, 1821-1828(1998).
  9. Basiouni, S., Fuhrmann, H. and Schumann, J., "High-efficiency Transfection of Suspension Cell Lines," Biotechniques 3, 1-4(2012).
  10. Shin, S.-E., Lim, J.-M., Koh, H. G., Kim, E. K., Kang, N. K., Jeon, S., Kwon, S., Shin, W.-S., Lee, B., Hwangbo, K., Kim, J., Ye, S. H., Yun, J.-Y., Seo, H., Oh, H.-M., Kim, K.-J., Kim, J.-S., Jeong, W.-J., Chang, Y. K. and Jeong, B.-R., "CRISPR/Cas9-induced Knockout and Knock-in Mutations in Chlamydomonas Reinhardtii," Sci. Rep.-UK 6, 27810(2016). https://doi.org/10.1038/srep27810
  11. Im, D. J., "Next Generation Digital Microfluidic Technology: Electrophoresis of Charged Droplets," Korean J. Chem. Eng., 32, 1001-1008(2015). https://doi.org/10.1007/s11814-015-0092-0
  12. Im, D. J., "Charging of an Ionic Liquid Droplet in a Dielectric Medium," Clean Technology 20, 354-358(2014). https://doi.org/10.7464/ksct.2014.20.4.354
  13. Im, D. J., Noh, J., Moon, D. and Kang, I. S. "Electrophoresis of a Charged Droplet in a Dielectric Liquid for Droplet Actuation," Anal. Chem., 83, 5168-5174(2011). https://doi.org/10.1021/ac200248x
  14. Im, D. J., Ahn, M. M., Yoo, B. S., Moon, D., Lee, D. W. and Kang, I. S. "Discrete Electrostatic Charge Transfer by the Electrophoresis of a Charged Droplet in a Dielectric Liquid," Langmuir 28, 11656-11661(2012). https://doi.org/10.1021/la3014392
  15. Im, D. J., Yoo, B. S., Ahn, M. M., Moon, D. and Kang, I. S., "Digital Electrophoresis of Charged Droplets," Anal. Chem., 85, 4038-4044(2013). https://doi.org/10.1021/ac303778j
  16. Ahn, M. M., Im, D. J. and Kang, I. S. "Geometric Characterization of Optimal Electrode Designs for Improved Droplet Charging and Actuation," Analyst 138, 7362-7368(2013). https://doi.org/10.1039/c3an01623d
  17. Lee, D. W., Im, D. J. and Kang, I. S., "Measurement of the Interfacial Tension in an Ionic Liquid-Dielectric Liquid System Using an Electrically Deformed Droplet," J. Phys. Chem. C., 117, 3426-3430(2013). https://doi.org/10.1021/jp312212e
  18. Ahn, M. M., Im, D. J., Kim, J. G., Lee, D. W. and Kang, I. S., "Extraction of Cations from an Ionic Liquid Droplet in a Dielectric Liquid under Electric Field," J. Phys. Chem. Lett., 5, 3021-3025(2014). https://doi.org/10.1021/jz501511z
  19. Ahn, M. M., Im, D. J., Yoo, B. S. and Kang, I. S., "Characterization of Electrode Alignment for Optimal Droplet Charging and Actuation in Droplet-based Microfluidic System," Electrophoresis 36, 2086-2093(2015). https://doi.org/10.1002/elps.201500141
  20. Choi, C. Y. and Im, D. J., "Contact Charging and Electrphoresis of a Glassy Carbon Microsphere," Korean Chem. Eng. Res., 54(4), 568-573(2016). https://doi.org/10.9713/kcer.2016.54.4.568
  21. Im, D. J., Jeong, S.-N., Yoo, B. S., Kim, B., Kim, D.-P., Jeong, W.-J. and Kang, I. S., "Digital Microfluidic Approach for Efficient Electroporation with High Productivity: Transgene Expression of Microalgae without Cell Wall Removal," Anal. Chem., 87, 6592-6599(2015). https://doi.org/10.1021/acs.analchem.5b00725
  22. Wang, S. N. and Lee, L. J., "Micro-/nanofluidics Based Cell Electroporation," Biomicrofluidics 7, 011301(2013). https://doi.org/10.1063/1.4774071
  23. Jung, J. H. and Lee, C. S. "Droplet Based Microfluidic System," Korean Chem. Eng. Res., 48(5), 545-555(2010).
  24. Im, D. J. and Jeong, S.-N. "Transfection of Jurkat T Cells by Droplet Electroporation," Biochem. Eng. J., 122, 133-140(2017). https://doi.org/10.1016/j.bej.2017.03.010
  25. Kurita, H., Takahashi, S., Asada, A., Matsuo, M., Kishikawa, K., Mizuno, A. and Numano, R., "Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor," PLOS ONE 10, e0144254(2015). https://doi.org/10.1371/journal.pone.0144254