JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Bioengenharia

使用无细胞系统对用于蛋白质原型的最小线性模板进行扩增的快速酶促方法

Published: June 14, 2021
doi:
10.3791/62728
,
1Chemical and Biological Engineering,Iowa State University

Summary

该研究描述了一种方案，用于在不克隆或使用活细胞的情况下从合成基因片段中创建大量（μg-mg）DNA以进行蛋白质筛选活动。将最小模板酶消化并循环化，然后使用等温滚动循环扩增进行扩增。无细胞表达反应可以用未纯化的产物进行。

Abstract

该协议描述了最小DNA模板的设计以及酶扩增的步骤，从而可以使用无细胞表达在不到24小时的时间内快速对可测定的蛋白质进行原型设计。从供应商处接收DNA后，对基因片段进行PCR扩增、切割、循环和冷冻储存。然后使用等温滚动圆扩增（RCA）稀释和显着扩增少量储存的DNA（高达106x）。RCA可以从起始物质的皮克水平（如果使用所有起始合成片段，则为mg水平）产生微克量的最小表达模板。在这项工作中，起始量为20 pg导致4μg的最终产物。所得的RCA产物（最小模板的串联体）可以直接添加到无细胞反应中，无需纯化步骤。由于这种方法完全基于PCR，因此当与自动化液体处理系统结合使用时，它可能会实现未来的高通量筛选工作。

Introduction

无细胞基因表达（CFE）已成为一种具有许多应用的强大工具。这些应用包括疾病检测1，2，3，4，5，6， 微量营养素和小分子检测7，8，9，10，11，12， 生物制造13，14，15，16，17 ，18， 教育19，20，21， 制造难解蛋白17，22，23，24，25，26，27， 和变异筛选23，28，29，30，31，32 ，33.这是由于无细胞系统的开放性和它们赋予的灵活性。许多伟大的评论文章提供了对技术的历史教育和未来展望34，35，36，37，38，39，40，41，42，43，44。

典型的无细胞反应由三个主要成分组成：细胞提取物，能量组合和遗传模板。活性细胞提取物包含转录和翻译（TXTL）所需的所有机器，并且可以以多种方式进行处理36。能量组合中的糖酵解中间体、电解质、氨基酸和辅因子支持 TXTL 过程。它是无细胞实验中变异性的主要来源45，并且可以以多种方式制备34，46。由于传统的克隆方法产生具有优异表达特性的质粒，因此遗传模板的制备得到了较少的改进。这些传统方法的缺点是构建和传播它们所需的周转时间和生物专业知识的数量。最近的优化工作已经为细胞提取物制备47，48提供了简单的24小时工作流程，可以与能量混合制备49，50并行进行。然而，传统的克隆为CFE原型设计时间增加了多天（表1）23。从商业基因片段中快速扩增的PCR产物可以直接使用51，但这限制了原型实验的数量，因为只产生1μgDNA，这对应于大约五个反应（传统的15μL体积）。通过这些额外的循环化和等温扩增步骤，DNA的量可能大于毫克（1毫克约5，000次反应）。这大大增加了在蛋白质或组合酶网络的高通量筛选（无细胞代谢工程）中可以进行的测试数量;它还允许将线性模板库有效保存为高浓度DNA。此外，需要增加模板的数量，以原型化材料科学应用所需的大量蛋白质（基于蛋白质的纤维和水凝胶）。线性模板的一些限制可以通过使用BL21 DE3 Star的提取物或使用最近发现的方法来保护线性模板免受退化52，53，54来克服。然而，这并不能解决供应商生产的用于PCR扩增的DNA库存有限或克隆所需的生物学专门知识和设备问题。

这项工作提出了一种方案，明确设计用于增加可以从供应商生产的少量基因片段（通常为500-1000ng冻干粉末）中获得的表达模板的数量。所描述的方法不需要在质粒中进行传统克隆或在活细胞中进行转化和繁殖所需的技能。在邮件中收到基因片段后，用户可以通过采用等温滚动圆扩增（RCA）（图1）23来产生足够的模板来进行许多无细胞反应。虽然从供应商处获得的DNA数量可能足以进行有限的筛选工作，但它很快就会耗尽，重新购买基因片段既耗时又昂贵。该方法也特别适用于大肠杆菌中有毒且难以克隆的基因。

Protocol

1. 设计基因片段 注意：基因片段应具有转录/翻译所需的所有遗传元件，包括启动子，核糖体结合位点（RBS），起始密码子，目的基因和终止子。虽然终止子对于线性表达模板（LET）不是必需的，但如果用户决定将序列插入质粒中，则这一点很重要。这些序列是从pJL1-sfGFP质粒55（ 来自Michael Jewett实验室的礼物）中取出的，该质粒使用T7启动子。除了这些必要的?…

Representative Results

当在15μL反应中仅使用0.30μL未纯化的RCA DNA时，RCA模板中sfGFP的表达与pJL1质粒的表达相当（图2A）。事实上，在BL21 DE3 Star提取物中，将模板的量增加一倍和三倍似乎没有益处，这表明模板的饱和水平已经达到每次反应0.30μL。相反，当添加到来自SHuffle菌株的细胞提取物中时，增加RCA模板的量似乎有好处（图2B）28。对于某些蛋白质，可以…

Discussion

目的基因可以是任何所需的蛋白质，但最好从荧光蛋白开始，作为方便的报告者，以便在孔板读数仪上进行实时或终点读数，以便新采用该方法。对于新的蛋白质序列，复制所需蛋白质的氨基酸序列并将其粘贴到所需的密码子优化工具61，62中。密码子优化工具中通常有许多可用的大肠杆菌生物和菌株，但选择一般大肠杆菌选项将是合适的?…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者承认NIH 1R35GM138265-01和NSF 2029532对该项目的部分支持。

Materials

Alaline Formedium DOC0102
Ammonium glutamate MP Biomedicals MP21805951
Arginine Formedium DOC0106
Asparagine Formedium DOC0114
Aspartic Acid Formedium DOC0118
ATP Sigma A2383
Axygen Sealing Film Corning PCR-SP
CMP Sigma C1006
Coenzyme A Sigma C3144
CutSmart Buffer NEB B7204S Provided with HindIII
Cysteine Formedium DOC0122
DNA Clean and Concentrator Kit Zymo Research D4004 Used for purifying DNA
dNTPs NEB N0447
E. coli tRNA Sigma (Roche) 10109541001
Folinic Acid Sigma 47612
Gene Fragment IDT
Glutamic Acid Formedium DOC0134
Glutamine Formedium DOC0130
Glycine Formedium DOC0138
GMP Sigma G8377
HEPES Sigma H3375
HindIII-HF NEB R3104L
Histidine Formedium DOC0142
Isoleucine Formedium DOC0150
Leucine Formedium DOC0154
Lysine Formedium DOC0158
Magnesium glutamate Sigma 49605
Methionine Formedium DOC0166
Microtiter Plate (384 well) Greiner 781906
Microtiter Plate (96 well) Greiner 655809
Multimode Plate Reader BioTek Synergy Neo2
NAD Sigma N8535
NanoPhotometer Implen NP80
OneTaq DNA Polymerase NEB M0480
PCR Tube VWR 20170-012
Phenylalanine Formedium DOC0170
Phosphoenolpyruvate Sigma (Roche) 10108294
Potassium glutamate Sigma G1501
Potassium oxalate Fisher Scientific P273
Proline Formedium DOC0174
Putrescine Sigma P5780
Serine Formedium DOC0178
Spermidine Sigma S0266
T4 DNA Ligase NEB M0202S
T4 DNA Ligase Reaction Buffer NEB B0202S Provided with T4 DNA Ligase
TempliPhi Amplification Kit Cytiva 25640010 Used for RCA
Thermal Cycler Biorad C1000 Touch
Thermoblock Eppendorf ThermoMixer FP
Threonine Formedium DOC0182
Tryptophan Formedium DOC0186
Tyrosine Formedium DOC0190
UMP Sigma U6375
Valine Formedium DOC0194
DOWNLOAD MATERIALS LIST

Referências

  1. Sun, Q., et al. A simple and low-cost paper-based colorimetric method for detecting and distinguishing the GII.4 and GII.17 genotypes of norovirus. Talanta. 225, 121978 (2021).
  2. Pardee, K., et al. Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 165 (5), 1255-1266 (2016).
  3. Pardee, K., et al. Paper-based synthetic gene networks. Cell. 159 (4), 940-954 (2014).
  4. Ma, D., Shen, L., Wu, K., Diehnelt, C. W., Green, A. A. Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment. Synthetic Biology. 3 (1), (2018).
  5. Park, S., Lee, J. W. Detection of coronaviruses using rna toehold switch sensors. International Journal of Molecular Sciences. 22 (4), 1772 (2021).
  6. Cao, M., Sun, Q., Zhang, X., Ma, Y., Wang, J. Detection and differentiation of respiratory syncytial virus subgroups A and B with colorimetric toehold switch sensors in a paper-based cell-free system. Biosensors and Bioelectronics. 182, 113173 (2021).
  7. Mcnerney, M. P., et al. Point-of-care biomarker quantification enabled by sample-specific calibration. Science Advances. 5 (9), (2019).
  8. Silverman, A. D., Akova, U., Alam, K. K., Jewett, M. C., Lucks, J. B. Design and optimization of a cell-free atrazine biosensor. ACS Synthetic Biology. 9 (3), 671-677 (2020).
  9. Salehi, A. S. M., et al. Cell-free protein synthesis approach to biosensing hTRβ-specific endocrine disruptors. Analytical Chemistry. 89 (6), 3395-3401 (2017).
  10. Garamella, J., Majumder, S., Liu, A. P., Noireaux, V. An adaptive synthetic cell based on mechanosensing, biosensing, and inducible gene circuits. ACS Synthetic Biology. 8 (8), 1913-1920 (2019).
  11. Glasscock, C. J., et al. Dynamic control of pathway expression with riboregulated switchable feedback promoters. ACS Synthetic Biology. 16, (2019).
  12. Thavarajah, W., et al. Point-of-use detection of environmental fluoride via a cell-free riboswitch-based biosensor. ACS Synthetic Biology. 9 (1), 10-18 (2020).
  13. Pardee, K., et al. Portable, on-demand biomolecular manufacturing. Cell. 167 (1), 248-259 (2016).
  14. Nelson, J. A. D., et al. Hydrofoam and oxygen headspace bioreactors improve cell-free therapeutic protein production yields through enhanced oxygen transport. Biotechnology Progress. 37 (2), 3079 (2020).
  15. Cai, Q., et al. A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems. Biotechnology Progress. 31 (3), 823-831 (2015).
  16. Ogonah, O. W., Polizzi, K. M., Bracewell, D. G. Cell free protein synthesis: a viable option for stratified medicines manufacturing? A brief history of cell free synthesis systems. Current Opinion in Chemical Engineering. 18, 77-83 (2017).
  17. Zawada, J. F., et al. Microscale to manufacturing scale-up of cell-free cytokine production-a new approach for shortening protein production development timelines. Biotechnology and Bioengineering. 108 (7), 1570-1578 (2011).
  18. Stark, J. C., et al. On-demand biomanufacturing of protective conjugate vaccines. Science Advances. 7 (6), (2021).
  19. Huang, A., et al. BioBitsTM Explorer: A modular synthetic biology education kit. Science Advances. 4 (8), 1-11 (2018).
  20. Stark, J. C., et al. BioBits health: classroom activities exploring engineering, biology, and human health with fluorescent readouts. ACS Synthetic Biology. 8 (5), 1001-1009 (2019).
  21. Stark, J. C., et al. BioBitsTM Bright: A fluorescent synthetic biology education kit. Science Advances. 4 (8), 33 (2018).
  22. Shinoda, T., et al. Cell-free methods to produce structurally intact mammalian membrane proteins. Scientific Reports. 6, (2016).
  23. Dopp, J. L., Rothstein, S. M., Mansell, T. J., Reuel, N. F. Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours. Biotechnology and Bioengineering. 116 (3), 667-676 (2019).
  24. Sachse, R., Dondapati, S. K., Fenz, S. F., Schmidt, T., Kubick, S. Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes. FEBS Letters. 588 (17), 2774-2781 (2014).
  25. Salehi, A. S. M., et al. Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system. Biotechnology Journal. 11 (2), 274-281 (2016).
  26. Georgi, V., et al. On-chip automation of cell-free protein synthesis: New opportunities due to a novel reaction mode. Lab on a Chip. 16 (2), 269-281 (2016).
  27. Thoring, L., et al. Cell-free systems based on CHO cell lysates: Optimization strategies, synthesis of “difficult-to-express” proteins and future perspectives. PLoS One. 11 (9), (2016).
  28. Dopp, J. L., Reuel, N. F. Simple, functional, inexpensive cell extract for in vitro prototyping of proteins with disulfide bonds. Biochemical Engineering Journal. 164, 107790 (2020).
  29. Isaksson, L., Enberg, J., Neutze, R., Göran Karlsson, B., Pedersen, A. Expression screening of membrane proteins with cell-free protein synthesis. Protein Expression and Purification. 82 (1), 218-225 (2012).
  30. Techner, J. M., et al. High-throughput synthesis and analysis of intact glycoproteins using SAMDI-MS. Analytical Chemistry. 92 (2), 1963-1971 (2020).
  31. Kim, H. C., et al. Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes. Biotechnology and Bioengineering. 112 (12), 2630-2635 (2015).
  32. Rolf, J., Siedentop, R., Lütz, S., Rosenthal, K. Screening and identification of novel cGAS homologues using a combination of in vitro and in vivo protein synthesis. International Journal of Molecular Sciences. 21 (1), (2020).
  33. Haslinger, K., Hackl, T., Prather, K. L. J. Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli. bioRxiv. , (2020).
  34. Dopp, J. L., Tamiev, D. D., Reuel, N. F. Cell-free supplement mixtures: Elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract. Biotechnology Advances. 37 (1), 246-258 (2018).
  35. Gregorio, N. E., Levine, M. Z., Oza, J. P. A user’s guide to cell-free protein synthesis. Methods and Protocols. 2 (1), 24 (2019).
  36. Cole, S. D., Miklos, A. E., Chiao, A. C., Sun, Z. Z., Lux, M. W. Methodologies for preparation of prokaryotic extracts for cell-free expression systems. Synthetic and Systems Biotechnology. 5 (4), 252-267 (2020).
  37. Chiba, C. H., Knirsch, M. C., Azzoni, A. R., Moreira, A. R., Stephano, M. A. Cell-free protein synthesis: advances on production process for biopharmaceuticals and immunobiological products. BioTechniques. 70, (2021).
  38. Laohakunakorn, N. Cell-free systems: A proving ground for rational biodesign. Frontiers in Bioengineering and Biotechnology. 8, 788 (2020).
  39. Dondapati, S. K., Stech, M., Zemella, A., Kubick, S. Cell-free protein synthesis: A promising option for future drug development. BioDrugs. , 1-22 (2020).
  40. Noireaux, V., Liu, A. P. The new age of cell-free biology. Annual Review of Biomedical Engineering. 22, 51-77 (2020).
  41. Khambhati, K., et al. Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems. Frontiers in Bioengineering and Biotechnology. 7, (2019).
  42. Carlson, E. D., Gan, R., Hodgman, C. E., Jewett, M. C. Cell-free protein synthesis: Applications come of age. Biotechnology Advances. 30 (5), 1185-1194 (2012).
  43. Rosenblum, G., Cooperman, B. S. Engine out of the chassis: Cell-free protein synthesis and its uses. FEBS Letters. 588 (2), 261-268 (2014).
  44. Swartz, J. R. Transforming biochemical engineering with cell-free biology. AIChE Journal. 58 (1), 5-13 (2012).
  45. Cole, S. D., et al. Quantification of interlaboratory cell-free protein synthesis variability. ACS Synthetic Biology. 8 (9), 2080-2091 (2019).
  46. Caschera, F., Noireaux, V. A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system. Metabolic Engineering. 27, 29-37 (2015).
  47. Levine, M. Z., et al. Activation of energy metabolism through growth media reformulation enables a 24-hour workflow for cell-free expression. ACS Synthetic Biology. 9 (10), 2765-2774 (2020).
  48. Hunt, J. P., et al. Streamlining the preparation of “endotoxin-free” ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase. Synthetic and Systems Biotechnology. 4 (4), 220-224 (2019).
  49. Dopp, J. L., Jo, Y. R., Reuel, N. F. Methods to reduce variability in E. Coli-based cell-free protein expression experiments. Synthetic and Systems Biotechnology. 4 (4), 204-211 (2019).
  50. Sun, Z. Z., et al. Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Journal of Visualized Experiments: JoVE. , e50762 (2013).
  51. Schinn, S. M., Broadbent, A., Bradley, W. T., Bundy, B. C. Protein synthesis directly from PCR: Progress and applications of cell-free protein synthesis with linear DNA. New Biotechnology. 33 (4), 480-487 (2016).
  52. Sitaraman, K., et al. A novel cell-free protein synthesis system. Journal of Biotechnology. 110 (3), 257-263 (2004).
  53. Marshall, R., Maxwell, C. S., Collins, S. P., Beisel, C. L., Noireaux, V. Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems. Biotechnology and Bioengineering. 114, 2137-2141 (2017).
  54. Sun, Z. Z., Yeung, E., Hayes, C. A., Noireaux, V., Murray, R. M. Linear DNA for rapid prototyping of synthetic biological circuits in an escherichia coli based TX-TL cell-free system. ACS Synthetic Biology. 3 (6), 387-397 (2014).
  55. . Addgene: pJL1 Available from: https://www.addgene.org/69496/ (2021)
  56. . IDT Codon Optimization Tool Available from: https://www.idtdna.com/pages/tools/codon-optimization-tool (2021)
  57. Hadi, T., et al. Rolling circle amplification of synthetic DNA accelerates biocatalytic determination of enzyme activity relative to conventional methods. Scientific Reports. 10 (1), 10279 (2020).
  58. . New England Biolabs Tm Calculator Available from: https://tmcalculator.neb.com/#!/main (2021)
  59. Shin, J., Noireaux, V. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70. Journal of Biological Engineering. 4, (2010).
  60. Colant, N., et al. A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions. Biotechnology Progress. 37 (1), 3062 (2021).
  61. Burgess-Brown, N. A., et al. Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study. Protein Expression and Purification. 59, 94-102 (2008).
  62. Maertens, B., et al. Gene optimization mechanisms: A multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli. Protein Science. 19 (7), 1312-1326 (2010).
  63. Eckert, K. A., Kunkel, T. A. DNA polymerase fidelity and the polymerase chain reaction. Genome Research. 1 (1), 17-24 (1991).
  64. Dopp, J. L., Reuel, N. F. Process optimization for scalable E. coli extract preparation for cell-free protein synthesis. Biochemical Engineering Journal. 138, 21-28 (2018).
  65. Liu, D. V., Zawada, J. F., Swartz, J. R. Streamlining Escherichia Coli S30 extract preparation for economical cell-free protein synthesis. Biotechnology Progress. 21 (2), 460-465 (2005).
  66. Levine, M. Z., Gregorio, N. E., Jewett, M. C., Watts, K. R., Oza, J. P. Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology. Journal of Visualized Experiments: JoVE. (144), e58882 (2019).
  67. Kwon, Y. C., Jewett, M. C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Scientific Reports. 5, (2015).

Tags

Keywords: Cell-free SystemsProtein PrototypingRolling Circle AmplificationPCRDNA TemplateRestriction DigestionDNA LigationEnzymatic Amplification
check_url/pt/62728?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Dopp, J. L., Reuel, N. F. Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems. J. Vis. Exp. (172), e62728, doi:10.3791/62728 (2021).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image