Optimization of GoldenBraid Digestion-ligation Reaction System

doi:10.13560/j.cnki.biotech.bull.1985.2020-0161

Abstract

Abstract: The objective of this work is to establish an efficient and stable GoldenBraid（GB）restriction-ligation assembly reaction system. In this study,enzyme digestion time,buffer system,restriction enzymes and ratio of substrates were optimized to improve the efficiency of effective cloning,and the optimal reaction system was determined. Results showed that the percentage of effective cloning was nearly 99% under 4 min enzyme digestion time,1 μL FastDigest Buffer（10×）buffer system,1 mmol/L ATP,0.5 μL FastDigest Esp3I or FastDigest Eco31I,and 3∶1 molar ratio of the inserts and acceptor plasmid in reaction system,which was over 4 times of that before optimization（BsmBI 8% and BsaI 23%）,and digestion-ligation efficiency was nearly 100%. The percentage of effective cloning significantly increased by optimizing reaction system even with less volume of restriction enzymes,compared to the original protocol with 1 μL restriction enzymes. This indicates that the efficiency and practicability of digestion-ligation in GB system may be improved via the optimized process.

Key words: GoldenBraid, optimization, effective cloning, efficiency of enzyme digestion and ligation

XU Mei-hui, ZHANG Yao-jie, TANG Ke-xuan, MIAO Zhi-qi. Optimization of GoldenBraid Digestion-ligation Reaction System[J]. Biotechnology Bulletin, 2020, 36(9): 266-274.

References

[1] Kiga D, Yamamura M.Synthetic biology[J]. New Generation Computing, 2008, 269(4):347-364.
[2] Bartley BA, Kim K, Medley JK, et al.Synthetic biology:Engineering living systems from biophysical principles[J]. Biophys J, 2017, 112(6):1050-1058.
[3] 肖文海, 王颖, 元英进. 化学品绿色制造核心技术造核心技术 1[J]. 化工学报, 2016, 67(1):119-128.
Xiao WH, Wang Y, Yuan JY.Core technology in chemicals green manufacturing:synthetic biology[J]. CIESC Journal, 2016, 67(1):119-128.
[4] 熊燕, 陈大明, 杨琛, 等. 合成生物学发展现状与前景[J]. 生命科学, 2011, 23(9):826-837.
Xiong Y, Chen DM, Yang C, et al.Progress and perspective of synthetic biology[J]. Chinese Bulletin of Life Sciences, 2011, 23(9):826-837.
[5] 朱星华, 李哲. 合成生物学的研究进展与应用[J]. 中国科技论坛, 2011(5):143-148.
Zhu XH, Li Z.The Research progress and future application of synthetic biology[J]. Forum on Science and Technology in China, 2011(5):143-148.
[6] Medema MH, Breitling R, Bovenberg R, et al.Exploiting plug-and-play synthetic biology for drug discovery and production in microorganisms[J]. Nat Rev Microbiol, 2011, 9(2):131-137.
[7] Breitling R, Takano E.Synthetic biology advances for pharmaceutical production[J]. Curr Opin Biotechnol, 2015, 35:46-51.
[8] 孔建强, 王伟, 程克棣, 等. 青蒿素的合成生物学研究进展[J]. 药学学报, 2013, 48(2):193-205.
Kong JQ, Wang W, Cheng KL, et al.Research progresses in synthetic biology of artemisinin[J]. Acta Pharmaceutica Sinica, 2013, 48(2):193-205.
[9] 蒙海林. 基于系统—合成生物学的天然产物异源生物合成研究[D]. 广州:华南理工大学, 2011.
Meng HL.Natural products heterologous biosynthesis based on Systems-synthetic biology[D]. Guangzhou:South China University of Technology, 2011.
[10] Jain KK, Synthetic biology and personalized medicine[J]. Med Princ Pract, 2013, 22(3):209-219.
[11] Ye H, Daoud-El Baba M, Peng RW, et al. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice[J]. Science, 2011, 332(6037):1565-1568.
[12] Weber W, Fussenegger M.Emerging biomedical applications of synthetic biology[J]. Nat Rev Gene, 2011, 13(1):21-35.
[13] Kemmer C, Fluri DA, Witschi U, et al.A designer network coordinating bovine artificial insemination by ovulation-triggered release of implanted sperms[J]. J Control Release, 2011, 150(1):23-29.
[14] Kemmer C, Gitzinger M, Daoud-El Baba M, et al. Self-sufficient control of urate homeostasis in mice by a synthetic circuit[J]. Nat Biotechnol, 2010, 28(4):355-360.
[15] Deans TL, Grainger DW, Fussenegger M.Synthetic Biology:Innovative approaches for pharmaceutics and drug delivery[J]. Adv Drug Deliv Rev, 2016, 105:1-2.
[16] Chao R, Yuan Y, Zhao H.Recent advances in DNA assembly technologies[J]. FEMS Yeast Res, 2015, 15(1):1-9.
[17] Casini A, Storch M, Baldwin GS, et al.Bricks and blueprints:methods and standards for DNA assembly[J]. Nat Rev Mol Cell Biol, 2015, 16(9):568-576.
[18] Cobb RE, Ning JC, Zhao H.DNA assembly techniques for next-generation combinatorial biosynthesis of natural products[J]. J Ind Microbiol Biotechnol, 2014, 41(2):469-477.
[19] Engler C, Marillonnet S.Generation of families of construct variants using golden gate shuffling[J]. Methods Mol Biol, 2011, 729:167-181.
[20] Gibson DG, Young L, Chuang RY, et al.Enzymatic assembly of DNA molecules up to several hundred kilobases[J]. Nat Methods, 2009, 6(5):343-345.
[21] Ellis T, Adie T, Baldwin GS.DNA assembly for synthetic biology:from parts to pathways and beyond[J]. Integr Biol, 2011, 3(2):109-118.
[22] 晁然, 原永波, 赵惠民. 构建合成生物学制造厂[J]. 中国科学:生命科学, 2015, 45(10):976-984.
Chao R, Yuan YB, Zhao HM.Building biological foundries for next generation synthetic biology[J]. Scientia Sinica(Vitae), 2015, 45(10):976-984.
[23] 常汉臣, 王琛, 王培霞, 等. DNA组装技术[J]. 生物工程学报, 2019, 35(12):2215-2226.
Chang HC, Wang C, Wang PX, et al.DNA assembly technologies:a review[J]. Chinese Journal of Biotechnology, 2019, 35(12):2215-2226.
[24] 张阳璞, 杨淑慎. 几种新型植物基因表达载体的构建方法[J]. 生物工程学报, 2015, 31(3):311-327.
Zhang YP, Yang SS.Methods for construction of transgenic plant expression vector:a review[J]. Chinese Journal of Biotechnology, 2015, 31(3):311-327.
[25] Sakuma T, Nishikawa A, Kume S, et al.Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system[J]. Sci Rep, 2014, 4:5400.
[26] Patron NJ.DNA assembly for plant biology[J]. Curr Protoc Plant Biol, 2016, 1(4):604-616.
[27] Sarrion-Perdigones A, Falconi EE, Zandalinas SI, et al.GoldenBraid:an iterative cloning system for standardized assembly of reusable genetic modules[J]. PLoS One, 2011, 6(7):e21622.
[28] Sarrion-Perdigones A, Vazquez-Vilar M, Palací J, et al.GoldenBraid 2. 0:a comprehensive DNA assembly framework for plant synthetic biology[J]. Plant Physiol, 2013, 162(3):1618-1631.
[29] Vazquez-Vilar M, Bernabé-Orts JM, Fernandez-Del-Carmen A, et al. A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard[J]. Plant Methods, 2016, 12:10.
[30] Bernabé-Orts JM, Casas-Rodrigo I, Minguet EG, et al.Assessment of Cas12a-mediated gene editing efficiency in plants[J]. Plant Biotechnol J, 2019, 17(10):1971-1984.
[31] Vazquez-Vilar M, Gandía M, García-Carpintero V, et al.Multigene engineering by GoldenBraid cloning:From plants to filamentous fungi and beyond[J]. Curr Protoc Mol Biol, 2020, 130(1):e116.
[32] Pérez-González A, Kniewel R, Veldhuizen M, et al.Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria[J]. BMC Biotechnol, 2017, 17(1):80.
[33] Hernanz-Koers M, Gandia M, Garrigues S, et al.FungalBraid:a GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology[J]. Fungal Genet Biol, 2018, 116:51-61.
[34] Vazquez-Vilar M, Quijano-Rubio A, Fernandez-Del-Carmen A, et al. GB3. 0:a platform for plant bio-design that connects functional DNA elements with associated biological data[J]. Nucleic Acids Res. 2017, 45(4):2196-2209.
[35] Sarrion-Perdigones A, Palaci J, Granell A, et al.Design and construction of multigenic constructs for plant biotechnology using the GoldenBraid cloning strategy[J]. Methods Mol Biol, 2014, 1116:133-151.
[36] 李建武, 李灏德, 文国宏, 等. 马铃薯EcoRⅠ/ MseⅠ内切酶组合 AFLP 反应体系的优化与引物筛选[J]. 江苏农业科学, 2017, 45(15):25-29.
Li JW, Li HD, Wen GH, et al.The potato EcoRⅠ/MseⅠ enzyme combination of AFLP reaction system optimization and primers screening[J]. Jiangsu Agricultural Sciences, 2017, 45(15):25-29.
[37] Wu AZ, Lin CZ, Zhai YJ, et al.Investigation of the interaction between two phenylethanoid glycosides and bovine serum albumin by spectroscopic methods[J]. J Pharm Anal, 2013, 3(1):61-65.
[38] 朱杭飞, 姜晓明, 藏雨轩, 等. 限制性内切酶KpnⅠ和EcoRⅠ双酶切条件的优化[J]. 吉林医药学院学报, 2014, 35(4):250-253.
Zhu KF, Jiang XM, Zang XY, et al.The optimization of co-digestion conditions by KpnⅠ /EcoR Ⅰ[J]. Journal of Jilin Medical University, 2014, 35(4):250-253.
[39] Plante D, Bélanger G, Leblanc D, et al.The use of bovine serum albumin to improve the RT-qPCR detection of foodborne viruses rinsed from vegetable[J]. Lett Appl Microbiol, 2011, 52(3):239-244.
[40] Wang T, Wang D, Lyu Y, et al.Construction of a high-efficiency cloning system using the Golden Gate method and I-SceI endonuclease for targeted gene replacement in Bacillus anthracis[J]. J Biotechnol, 2018, 271:8-16.
[41] Li B, Kaushik S, Kalinowski P, et al.Droplet digital PCR shows the D-Loop to be an error prone locus for mitochondrial DNA copy number determination[J]. Sci Rep, 2018, 8(1):11392.
[42] 付峰. 鲤春病毒血症病毒(SVCV)糖蛋白基因分析、改造及其克隆与表达[D]. 上海:上海海洋大学, 2005.
Fu F.Bioinformatic analysis, modification, cloning, and expression of the glycoprotein gene of spring viremia of carp virus(SVCV)[D]. Shanghai:Shanghai Ocean University, 2005.