디지털 전기천공을 이용한 미세조류 내 단백질 전달 연구

임도진; Do Jin Im

Korean Chemical Engineering Research, Vol.56, No.1, 79-84, February, 2018

DOI10.9713/kcer.2018.56.1.79 Export Citation

디지털 전기천공을 이용한 미세조류 내 단백질 전달 연구

Delivery of Protein into Microalgae by the Digital Electroporation

임도진^†

국립부경대학교 화학공학과, 48547 부산광역시 남구 신선로 365

Do Jin Im^†

Department of Chemical Engineering, Pukyong National University, 365, Sinseon-ro, Nam-gu, Busan, 48547, Korea

E-mail:

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 세포의핵 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 특성분석 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.

Keywords:Digital electroporation;Microalgae;Protein delivery;CRISPR/CAS9

[References]

Wijffels RH, Barbosa MJ, Science, 329(5993), 796 (2010)
Pires JCM, Alvim-Ferraz MCM, Martins FG, Simoes M, Renew. Sust. Energ. Rev., 16, 3043 (2012)
Guo SL, Zhao XQ, Tang Y, Wan C, Alam MA, Ho SH, Bai FW, Chang JS, J. Biotechnol., 163, 61 (2013)
Gimpel JA, Specht EA, Georgianna DR, Mayfield SP, Curr. Opin. Chem. Biol., 17, 489 (2013)
Scranton MA, Ostrand JT, Fields FJ, Mayfield SP, Plant J., 82, 523 (2015)
Specht EA, Miyake-Stoner S, Mayfield SP, Biotech. Lett., 32, 1373 (2010)
Jo JM, Shin SG, Jung HJ, Min BR, Kim SK, Kim JW, Korean Chem. Eng. Res., 55(4), 542 (2017)
Shimogawara K, Fujiwara S, Grossman A, Usuda H, Genetics, 148, 1821 (1998)
Basiouni S, Fuhrmann H, Schumann J, Biotechniques, 3, 1 (2012)
Shin SE, Lim JM, Koh HG, Kim EK, Kang NK, Jeon S, Kwon S, Shin WS, et al., Sci. Rep.-UK, 6, 27810 (2016)
Im DJ, Korean J. Chem. Eng., 32(6), 1001 (2015)
Im DJ, Clean Technol., 20(4), 354 (2014)
Im DJ, Noh J, Moon D, Kang IS, Anal. Chem., 83, 5168 (2011)
Im DJ, Ahn MM, Yoo BS, Moon D, Lee DW, Kang IS, Langmuir, 28(32), 11656 (2012)
Im DJ, Yoo BS, Ahn MM, Moon D, Kang IS, Anal. Chem., 85, 4038 (2013)
Ahn MM, Im DJ, Kang IS, Analyst, 138, 7362 (2013)
Lee DW, Im DJ, Kang IS, J. Phys. Chem., 117, 3426 (2013)
Ahn MM, Im DJ, Kim JG, Lee DW, Kang IS, J. Phys. Chem. Lett., 5, 3021 (2014)
Ahn MM, Im DJ, Yoo BS, Kang IS, Electrophoresis, 36(17), 2086 (2015)
Choi CY, Im DJ, Korean Chem. Eng. Res., 54(4), 568 (2016)
Im DJ, Jeong SN, Yoo BS, Kim B, Kim DP, Jeong WJ, Kang IS, Anal. Chem., 87, 6592 (2015)
Wang SN, Lee LJ, Biomicrofluidics, 7, 011301 (2013)
Jung JH, Lee CS, Korean Chem. Eng. Res., 48(5), 545 (2010)
Im DJ, Jeong SN, Biochem. Eng. J., 122, 133 (2017)
Kurita H, Takahashi S, Asada A, Matsuo M, Kishikawa K, Mizuno A, Numano R, PLOS ONE, 10, e01442 (2015)

[Referenced By]

[1] Wijffels RH, Barbosa MJ, Science, 329(5993), 796 (2010)

[2] Pires JCM, Alvim-Ferraz MCM, Martins FG, Simoes M, Renew. Sust. Energ. Rev., 16, 3043 (2012)

[3] Guo SL, Zhao XQ, Tang Y, Wan C, Alam MA, Ho SH, Bai FW, Chang JS, J. Biotechnol., 163, 61 (2013)

[4] Gimpel JA, Specht EA, Georgianna DR, Mayfield SP, Curr. Opin. Chem. Biol., 17, 489 (2013)

[5] Scranton MA, Ostrand JT, Fields FJ, Mayfield SP, Plant J., 82, 523 (2015)

[6] Specht EA, Miyake-Stoner S, Mayfield SP, Biotech. Lett., 32, 1373 (2010)

[7] Jo JM, Shin SG, Jung HJ, Min BR, Kim SK, Kim JW, Korean Chem. Eng. Res., 55(4), 542 (2017)

[8] Shimogawara K, Fujiwara S, Grossman A, Usuda H, Genetics, 148, 1821 (1998)

[9] Basiouni S, Fuhrmann H, Schumann J, Biotechniques, 3, 1 (2012)

[10] Shin SE, Lim JM, Koh HG, Kim EK, Kang NK, Jeon S, Kwon S, Shin WS, et al., Sci. Rep.-UK, 6, 27810 (2016)

[11] Im DJ, Korean J. Chem. Eng., 32(6), 1001 (2015)

[12] Im DJ, Clean Technol., 20(4), 354 (2014)

[13] Im DJ, Noh J, Moon D, Kang IS, Anal. Chem., 83, 5168 (2011)

[14] Im DJ, Ahn MM, Yoo BS, Moon D, Lee DW, Kang IS, Langmuir, 28(32), 11656 (2012)

[15] Im DJ, Yoo BS, Ahn MM, Moon D, Kang IS, Anal. Chem., 85, 4038 (2013)

[16] Ahn MM, Im DJ, Kang IS, Analyst, 138, 7362 (2013)

[17] Lee DW, Im DJ, Kang IS, J. Phys. Chem., 117, 3426 (2013)

[18] Ahn MM, Im DJ, Kim JG, Lee DW, Kang IS, J. Phys. Chem. Lett., 5, 3021 (2014)

[19] Ahn MM, Im DJ, Yoo BS, Kang IS, Electrophoresis, 36(17), 2086 (2015)

[20] Choi CY, Im DJ, Korean Chem. Eng. Res., 54(4), 568 (2016)

[21] Im DJ, Jeong SN, Yoo BS, Kim B, Kim DP, Jeong WJ, Kang IS, Anal. Chem., 87, 6592 (2015)

[22] Wang SN, Lee LJ, Biomicrofluidics, 7, 011301 (2013)

[23] Jung JH, Lee CS, Korean Chem. Eng. Res., 48(5), 545 (2010)

[24] Im DJ, Jeong SN, Biochem. Eng. J., 122, 133 (2017)

[25] Kurita H, Takahashi S, Asada A, Matsuo M, Kishikawa K, Mizuno A, Numano R, PLOS ONE, 10, e01442 (2015)