我们感谢 Swapnil 巴蒂亚, 亚历杭德佩洛斯, 和约翰逊林为木偶工程的工作, 以及 Swati 卡尔, 雷切尔, 史密斯和托马斯在这份手稿的帮助。这项工作由 NSF 职业奖 #1253856 资助。它还由 NSF #1522074 的计算奖资助。

1. 指定在 DNA 设备库中使用的部件并生成用户/液体处理程序指令 [15 分钟]
<ol><li>使用任何 web 浏览器, 导航到 mocloassembly.com 和上传基因库文件的所有 dna 部分将包括在组合 dna 装置设计。</li>
	<li>一旦所有文件都被上传, 选择所需的 dna 部件, 并将它们拖到空白画布上, 将部分类型放置在预期的 dna 部分的最终顺序中。 注意: 可以选择零件的集合以及单个零件, 并将其放置在画布上。此外, 命令 DNA 部分, 使 5 ' 和 3 ' 悬垂每个部分匹配。</li>
	<li>单击页面右下角的 "组合"。 注意: 该工具将只产生有效的, 可的组件基于四基对悬垂每个部分后, 消化与 BsaI 酶。如果没有基于科学家上传的部件的可 DNA 设备, 该工具将指示没有找到任何程序集。</li>
	<li>
导航到 "计划" 选项卡并下载由该工具生成的文件。这些文件将包括:
	<ol>
<li>人可读的板材地图为科学家准备脱氧核糖核酸样品, 并且试剂必要为反应</li>
		<li>液体处理程序的 "选择列表"</li>
		<li>所有要组装的 DNA 设备的完整标注的基因库文件 注: 在进入任何新的实验室时, 液体搬运臂的定位必须经过测试。有关如何根据需要调整 X、Y 和 Z 轴的移臂定位的详细说明, 请参阅制造商手册。</li>
	</ol>
</li>
</ol>2. 制备组装的质粒 DNA 和试剂 [3 天]
<ol><li>[1 天]使用无菌接种回路和工作在开放的火焰附近或在层流罩, 条纹出细菌甘油库存到 LB 琼脂板补充适当的抗生素。为所有必要的 DNA 部件做这件事, 确保在每个样品之间进行循环消毒。在37° c 的夜间孵育板。 注: 冰冻细菌甘油库存应尽可能保持在冰上。反复冻融循环降低了库存的可行性, 应避免。</li>
	<li>[2 天]使用无菌吸管尖端, 牙签, 或接种回路, 并在开放的火焰或层流罩工作, 接种3毫升的 lb 肉汤 (辅以适当的抗生素) 与一个单一的殖民地从 lb 琼脂板准备在2.1。在37° c 的夜间孵育培养基, 同时在 300 RPM 晃动。</li>
	<li>[3 天]利用任何商业上可用的微质粒纯化试剂盒净化细菌培养物中的质粒 DNA。</li>
	<li>使用所提供的 MoClo_Setup xlsx 文件, 将质粒 DNA 的每个样本稀释到 20 fmol/µL 在水或 TE 缓冲液中的浓度。</li>
	<li>按照组装工具生成的 PDF 文件的板图, 将每个稀释的 DNA 部分的指示体积放在一个完全掠过的96井 PCR 板上的适当井中。把这个 SetupPlate 在冰上, 直到需要, 或密封与铝箔胶粘剂密封和存储在-20 ° c。</li>
	<li>
在冰上, 准备反应 mastermix 与以下组分: 为每20µL 反应增加2µL 10x T4 脱氧核糖核酸连接缓冲, 0.5 µL 脱氧核糖核酸 T4 (HC) 和1连接µL 酵素。MoClo_Setup. xlsx 文件中包含一张计算器表, 以协助处理此事。
	<ol>
<li>将酶 mastermix 到新的全掠过的96阱 PCR 板的适当井中, 在生成的 PDF 中跟随 ReagentPlate 的板映射。把这个 ReagentPlate 在冰上或96井冷块上。 注意: ReagentPlate 只应准备好在液体处理程序上运行程序集。</li>
	</ol>
</li>
</ol>3. 在液体处理程序上执行装配脚本 [变量]
<ol><li>将 SetupPlate (s), ReagentPlate (s) (在一个96井的冷块), 和必要数量的空全掠过 96-良好的 PCR 板在甲板上的液体处理。空板 (s) 将是 OutputPlate (s) 的反应组装。</li>
	<li>准备液体处理程序控制软件通过创建每个样品和试剂板的实例准备, 确保他们的名字完全一样, 因为它们出现在 mocloassembly.com 产生的板块地图, 包括一槽清洁, 去离子水标记水库 "。</li>
	<li>使用控制软件中的 "工作" 命令, 加载由我们的软件工具生成的. 长城文件, 后跟另一个 "工作" 命令, 它将执行在第一个命令中加载的. 长城文件。</li>
	<li>使用控制器软件的 "Run" 命令执行脚本。 注意: 在尝试访问甲板空间之前, 始终允许机器人液体处理程序完成脚本的执行。</li>
	<li>从液体处理甲板上卸下所有板。剩余的 DNA 可以通过密封 SetupPlate (s) 与铝密封膜和存储在-20 ° c。密封 OutputPlate (s) 与胶膜, 放置在一个 thermocycler 或热块, 并运行以下周期参数: 37° c 为 2 h, 50 ° c 为 5 min, 80 ° c 为 10 min, 举行在4° c 注: 一旦反应冷热完成, OutputPlate (s) 可以储存在-20 ° c, 直到他们准备好要转换。</li>
</ol>4. 转换反应 [1 天]
<ol><li>
解冻必要数量的合格的大肠杆菌细胞等分 (10 µL/反应) 冰。 注意: 为了保持不育, 以下步骤应在明火附近或层流罩内进行。<ol>
<li>当细胞解冻时, 用移50µL 0.1 米 IPTG 和50µL 20 毫克/毫升 X 加仑在表面上制备 LB 琼脂 (含有适当的抗生素)。如果电镀大量的反应, 做一个主混合。用无菌的玻璃棒或玻璃珠均匀地涂上盘子, 让盘子在37° c 下休息至少15分钟才能镀细菌。 注: 或者, 将 IPTG 和 x gal 添加到液体琼脂前, 在盘子被浇, 在2毫米 IPTG 和40µg/毫升 x 加仑最终浓度。存储这些板块的直接光, 因为 X Gal 是轻敏感。</li>
	</ol>
</li>
	<li>在冰上, 分10µL 的合格细胞为每个反应进入一个新的 96-井 PCR 板。这将是转换板。</li>
	<li>从 OutputPlate (s) 向相应的井中添加1-3 µL, 并在冰上孵育5分钟。</li>
	<li>在 thermocycler 42 ° c 的三十年代, 用粘合剂膜和热冲击密封转换板。然后, 立即把盘子放在冰上2分钟。</li>
	<li>对同一个井的转换板, 添加150µL 的 SOC 介质, 密封与铝胶粘剂密封, 并孵化在37° c, 而颤抖 900 RPM 为1小时。</li>
	<li>板的全部内容的转换板上的 LB 琼脂板上的4.1.1 使用无菌玻璃棒或玻璃珠均匀涂层表面的板。在37° c 的夜间孵育板。</li>
</ol>5. 克隆人验证 [2 天]
<ol><li>
用1.5 毫升的 LB 肉汤 (含适当的抗生素) 准备一个或几个96井深井培养块。
	<ol>
<li>与步骤2.2 相似, 将深井培养块与单个白色菌落从每一个 LB 琼脂的转化反应板中接种。</li>
		<li>密封的文化块 (s) 与透气密封和孵化过夜在37° c, 而颤抖 900 RPM。 注: 模块化克隆技术采用蓝白筛选, 所以阳性 CFUs 将出现白色的 LB 琼脂板, 而空的目标向量将出现蓝色。</li>
	</ol>
</li>
	<li>分离细菌培养物中的质粒 DNA (如 2.3), 并提交桑格测序以验证克隆。</li>
</ol>

<ol>
	<li>Gardner, T. S., Cantor, C. R., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+genetic+toggle+switch+in+Escherichia+coli.">Construction of a genetic toggle switch in Escherichia coli.</a> Nature. 403 (6767), 339-342 (2000).</li><li>Elowitz, M. B., Leibler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+oscillatory+network+of+transcriptional+regulators.">A synthetic oscillatory network of transcriptional regulators.</a> Nature. 403 (6767), 335-338 (2000).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nat Biotechnol. 23 (1), 47-55 (2005).</li><li>Anderson, J. C., Clarke, E. J., Arkin, A. P., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmentally+controlled+invasion+of+cancer+cells+by+engineered+bacteria.">Environmentally controlled invasion of cancer cells by engineered bacteria.</a> J Mol Biol. 355 (4), 619-627 (2006).</li><li>Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R., Benenson, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-input+RNAi-based+logic+circuit+for+identification+of+specific+cancer+cells.">Multi-input RNAi-based logic circuit for identification of specific cancer cells.</a> Science. 333 (6047), 1307-1311 (2011).</li><li>Ro, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+the+antimalarial+drug+precursor+artemisinic+acid+in+engineered+yeast.">Production of the antimalarial drug precursor artemisinic acid in engineered yeast.</a> Nature. 440 (7086), 940-943 (2006).</li><li>Georgianna, D. R., Mayfield, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+diversity+and+synthetic+biology+for+the+production+of+algal+biofuels.">Exploiting diversity and synthetic biology for the production of algal biofuels.</a> Nature. 488 (7411), 329-335 (2012).</li><li>Savage, D. F., Way, J., Silver, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defossiling+fuel:+how+synthetic+biology+can+transform+biofuel+production.">Defossiling fuel: how synthetic biology can transform biofuel production.</a> ACS Chem Biol. 3 (1), 13-16 (2008).</li><li>Fussenegger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptogramin-based+gene+regulation+systems+for+mammalian+cells.">Streptogramin-based gene regulation systems for mammalian cells.</a> Nat Biotechnol. 18 (11), 1203-1208 (2000).</li><li>Boorsma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+temperature-regulated+replicon-based+DNA+expression+system.">A temperature-regulated replicon-based DNA expression system.</a> Nat Biotechnol. 18 (4), 429-432 (2000).</li><li>Malphettes, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mammalian+expression+system+derived+from+components+coordinating+nicotine+degradation+in+arthrobacter+nicotinovorans+pAO1.">A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1.</a> Nucleic Acids Res. 33 (12), e107 (2005).</li><li>Brophy, J. A., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+genetic+circuit+design.">Principles of genetic circuit design.</a> Nat Methods. 11 (5), 508-520 (2014).</li><li>Lou, C., Stanton, B., Chen, Y. J., Munsky, B., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribozyme-based+insulator+parts+buffer+synthetic+circuits+from+genetic+context.">Ribozyme-based insulator parts buffer synthetic circuits from genetic context.</a> Nat Biotechnol. , (2012).</li><li>Cardinale, S., Arkin, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contextualizing+context+for+synthetic+biology--identifying+causes+of+failure+of+synthetic+biological+systems.">Contextualizing context for synthetic biology--identifying causes of failure of synthetic biological systems.</a> Biotechnol J. 7 (7), 856-866 (2012).</li><li>Carr, S. B., Beal, J., Densmore, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+DNA+context+dependence+in+bacterial+promoters.">Reducing DNA context dependence in bacterial promoters.</a> PLoS One. 12 (4), e0176013 (2017).</li><li>Yeung, E., Ng, A., Kim, J., Sun, Z. Z., Murray, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Decision and Control (CDC), 2014 IEEE 53rd Annual Conference on. , 5405-5412 (2014).</li><li>Engler, C., Gruetzner, R., Kandzia, R., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golden+gate+shuffling:+a+one-pot+DNA+shuffling+method+based+on+type+IIs+restriction+enzymes.">Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.</a> PLoS One. 4 (5), e5553 (2009).</li><li>Weber, E., Engler, C., Gruetzner, R., Werner, S., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+cloning+system+for+standardized+assembly+of+multigene+constructs.">A modular cloning system for standardized assembly of multigene constructs.</a> PLoS One. 6 (2), e16765 (2011).</li><li>Iverson, S. V., Haddock, T. L., Beal, J., Densmore, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIDAR+MoClo:+Improved+MoClo+Assembly+Standard+and+New+E.+coli+Part+Library+Enable+Rapid+Combinatorial+Design+for+Synthetic+and+Traditional+Biology.">CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology.</a> ACS Synth Biol. 5 (1), 99-103 (2016).</li><li>Casini, A., Storch, M., Baldwin, G. S., Ellis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bricks+and+blueprints:+methods+and+standards+for+DNA+assembly.">Bricks and blueprints: methods and standards for DNA assembly.</a> Nat Rev Mol Cell Biol. 16 (9), 568-576 (2015).</li><li>Bhatia, S. P., Smanski, M., Voigt, C. A., Densmore, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+design+via+combinatorial+constraint+specification.">Genetic design via combinatorial constraint specification.</a> ACS Synth Biol. , (2017).</li><li>Ham, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+implementation+and+practice+of+JBEI-ICE:+an+open+source+biological+part+registry+platform+and+tools.">Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools.</a> Nucleic Acids Res. 40 (18), e141 (2012).</li><li>Madsen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SBOL+Stack:+A+Platform+for+Storing,+Publishing,+and+Sharing+Synthetic+Biology+Designs.">The SBOL Stack: A Platform for Storing, Publishing, and Sharing Synthetic Biology Designs.</a> ACS Synth Biol. 5 (6), 487-497 (2016).</li><li>Knight, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Draft standard for BioBrick biological parts. , (2007).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Ma, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FusX:+A+Rapid+One-Step+Transcription+Activator-Like+Effector+Assembly+System+for+Genome+Science.">FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science.</a> Hum Gene Ther. 27 (6), 451-463 (2016).</li><li>Check Hayden, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+automated+lab.">The automated lab.</a> Nature. 516 (7529), 131-132 (2014).</li><li>Nielsen, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+circuit+design+automation.">Genetic circuit design automation.</a> Science. 352 (6281), aac7341 (2016).</li><li>Woodruff, L. B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registry+in+a+tube:+multiplexed+pools+of+retrievable+parts+for+genetic+design+space+exploration.">Registry in a tube: multiplexed pools of retrievable parts for genetic design space exploration.</a> Nucleic Acids Res. 45 (3), 1553-1565 (2017).</li><li>Hasty, J., McMillen, D., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+gene+circuits.">Engineered gene circuits.</a> Nature. 420 (6912), 224-230 (2002).</li><li>Gibson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+overlapping+DNA+fragments.">Enzymatic assembly of overlapping DNA fragments.</a> Methods Enzymol. 498, 349-361 (2011).</li><li>Appleton, E., Tao, J., Haddock, T., Densmore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactive+assembly+algorithms+for+molecular+cloning.">Interactive assembly algorithms for molecular cloning.</a> Nat Methods. 11 (6), 657-662 (2014).</li><li>Vasilev, V., Liu, C., Haddock, T., Bhatia, S., Adler, A., Yaman, F., Beal, J., Babb, J., Weiss, R., Densmore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Software+Stack+for+Specification+and+Robotic+Execution+of+Protocols+for+Synthetic+Biological+Engineering.">A Software Stack for Specification and Robotic Execution of Protocols for Synthetic Biological Engineering.</a> SynBERC Fall Retreat, Harvard University. , (2011).</li><li>Beal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+end-to-end+workflow+for+engineering+of+biological+networks+from+high-level+specifications.">An end-to-end workflow for engineering of biological networks from high-level specifications.</a> ACS Synth Biol. 1 (8), 317-331 (2012).</li><li>Bhatia, S., Densmore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigeon:+a+design+visualizer+for+synthetic+biology.">Pigeon: a design visualizer for synthetic biology.</a> ACS Synth Biol. 2 (6), 348-350 (2013).</li></ol>

Densmore 是总统和晶格自动化的共同创始人, Inc. 提蒙斯和麦卡锡是格子雇员。格子使软件和服务为自动化的许多过程描述了。

总之, 完整的 DNA 设备创建过程的自动化, 从在硅片中设计到液体处理, 是目前现有技术的一个可行的目标。软件和现代机器人技术允许创建成本效率高、时间效率高且可伸缩的工作流, 同时还可生成比手动方法更一致的可重现结果。尽管自动化不一定是执行协议的最经济有效的选择, 但它确实提高了实验的重复性, 并释放了宝贵的研究员时间。然而, 根据所使用的硬件, 自动化有时会使成本和执行时间远远低于通过常规手工方法所能达到的水平。此外, 自动化以形式化的方式捕获协议, 以防止即席、手工和轶事的最佳实践实验。这里展示了模块化 DNA 设备的自动装配, 并提供了读取器执行这些功能所需的协议、电子文件和物理 dna 资源, 以及类似的实验。我们希望我们的工具的可用性, 以及本议定书的出版将作为一种资源, 并将该领域推向更透明和共同的未来, 在 DNA 组装过程和液体处理机器人领域。
自动化硬件的用处在很大程度上取决于硬件的配置 & 功能。例如, 我们的液体处理器使用系统流体置换来驱动吸气和免除指令。驱动系统流体的活塞是相对较大的1毫升注射器, 虽然对一系列的体积来说很有用, 但对精确的试剂分配施加了2µL 下限。因此, 我们扩大了在液体处理程序上建立的克隆反应的总体积到20µL, 因为每个分配命令都需要≥2µL。这有效地将每反应的成本提高了一倍, 为液体处理剂准备的反应, 但是执行这些反应所需的手的时间的数量大大减少。为了解决这个问题, 我们重复了在声学液体分配器上的所有96反应的反应设置。该装置使用声音能量将流体直接从一个板块分配到另一板, 并可以实现分配量远远低于 (2.5 nL) 什么是可能的标准空气置换的手动管。使用这个装置, 我们可以缩小我们的反应总量为 250 nL, 40 倍的减少相比, 手工准备的反应10µL。由于小批量配发, 并缺乏小费之间的变化移步骤, 声学分配器能够产生相同的96反应在一小部分时间 (< 5 分钟)。较小的反应量也节省了废弃的试剂, 因为我们通常只转换1-3 µL 的反应。这样说, 自动化硬件的使用, 加上直观的软件, 可以使大量的 DNA 组装反应的一代更广泛的学术受众。
在这里, 我们展示了我们的软件工具的效用, 但有许多功能, 将有助于扩大其实用性。首先, 每次使用该工具来生成组合组件时, 必须生成新的 DNA 部件板, 要求科学家手动填充这些板以供每个组装运行。相反, 如果科学家能够指定在组装中使用的 DNA 板块中的零件位置, 那将是有帮助的。这将允许使用高通量质粒 DNA 纯化试剂盒, 因为研究人员可以从 CIDAR MoClo 库这样的试剂盒中接种培养物, 并将所有样品一起纯化, 同时保持试剂盒中所指定的每个部件的良好位置。第二, 该工具目前只支持使用96井板。对于需要建立数百个 dna 装置的大型项目, dna、试剂和目标板的数量可能超过液体处理器的甲板容量。这一问题至少部分地可以通过支持高密度板格式 (384 或 1536-井) 得到缓解。最后, 该工具目前只支持单一类型的液体处理和 DNA 组装策略。虽然使用电子表格软件将工具生成的液体处理指令转换为声学分配器格式相对容易, 但我们希望将本地支持扩展到许多不同的液体处理程序, 从而大大拓宽其适用性,将与其他常见的 DNA 组装技术如吉布森汇编31兼容。
这一自动化组件生态系统的一个关键部分是一套软件工具, 它将高级装配计划转换成自动化友好协议, 并明确计划在液体搬运机器人上运行。虽然存在一些软件工具, 允许研究人员设计在硅片中的程序集, 包括 Benchling、MoClo 规划器和乌鸦32, 但很少有能力将这些设计转换成可执行指令, 以在液体上运行处理.为此, 公关自动化和木偶操纵等工作33,34,35已经开始使这些工具可用。此外, 在这一领域工作的商业实体正在想办法引入 "云实验室", 通过自动化向大型终端用户群体提供实验服务。本文件概述的议定书可以作为任何这些努力的一部分, 只要它们作为一种服务提出。
虽然自动组装的 DNA 设备是直接和明显的价值, 以合成生物学, 我们的协议是有用的更大的群体的分子生物学家以及。自动化 DNA 组装允许大量的已知的, 但相似的, 基因设备的并行创建, 并能使表达库的快速合成为筛选和测试目的的药物开发研究。我们希望, 我们的软件工具将使更大的组合基的 DNA 组装工作更容易接近, 并作为一个有用的资源, 无论是合成生物学, 以及更大的学术团体。

最早的合成生物遗传设备, 如柯林斯切换开关1和 Elowitz repressilator2表明, 生物系统可以被设计为具有特定的确定性功能。从那时起, 合成生物学家就在生物材料的服务中努力设计生活系统, 以逐步实现更复杂的功能3, biotherapeutics4,5,6, 生物燃料7、8和传感应用程序91011。通过将模块化 DNA "部件" 与特定功能结合成 "设备" 来实现这些应用, 是合成生物学的主要目标之一。要使这一过程扩展, 必须有一种技术, 允许从大型的部件库中创建复杂的设备, 并以高效、经济高效、最重要的方式重现。
这样一个膨胀的装配过程是必要的, 因为目前这个领域缺乏对指导成功的生物系统设计和组成的规则的完全的理解。这是由于没有充分特征的 DNA 部件12, 缺乏兼容性和组合部件13, 以及在合成设备中的遗传组件之间的意外的, 不必要的交互而加剧14,15. 在缺乏可靠的预测模型的情况下, 功能性的合成遗传设备是通过试验和误差来达到的, 这就要求对预期设备的输入输出信号强度变量进行数十个甚至数百种筛选, 并进行 "最佳"为具有下游元素16的组合选择。虽然现代标准化的 DNA 组装方法, 如金门17和模块化克隆18,19,20 , 使这个过程更容易, 仍然需要一个实验专家来执行每个协议。随着合成设备的大小和复杂性的增长, 总可用的设计空间将变得太大, 无法手动构造和测试21, 而且该过程对于在该字段中可复制的任何重大进展都将过于手工操作。
直到合成生物学和生物部分存储库 (如 iGEM 部件注册表 (http://partsregistry.org)、组合元素的 JBEI 清单22和 SynBioHub23的出现, 基因部件才存储在任何标准化的程序集格式。每个项目只需要克隆少量的部件, 因此, 克隆所做的数量很少, 而与实际的研究目标相比, 组装设备的实现是可行的, 而且微不足道。分子克隆通常是ad hoc , 使用基于限制站点和切可用性的限制摘要执行, 而不是遵循任何标准化的过程。由于缺乏标准化, 使任何克隆协议自动化变得不切实际, 因为下一个克隆反应不可能遵循相同的协议。此外, 自动化 DNA 组装需要大量的货币投资设备 (液体搬运机器人及其相关软件和实验室基础设施), 以及时间投资的指示, 以发展准确处理所处理的各种液体的参数和运行这些协议的精确的指令系列。小规模的克隆努力并不能证明这些开支是合理的。更大, 更复杂的遗传设备设计结合标准化的组装协议24,25创建了一个环境, 其中自动化这些过程是非常实际的。低成本的机器人, 如 Opentrons OT-一个26也正在出现, 它允许即使是适度资助的实验室也能访问这种技术。此外, "云" 实验室27 , 包括 Transcriptic 和翡翠云实验室, 以及学术 "biofoundries", 如爱丁堡基因组铸造, UIUC iBioFab, 和麻省理工学院的广泛铸造, 利用机器人组装各种设计为各种客户快速和重复, 同时维护一个基本的 DNA 基元和组装技术的未来订单的共同存储库。
在自动化 DNA 组装过程中最大的挑战之一是为液体处理程序生成移命令。虽然这些设备的软件接口通常很容易使用, 但复杂的移指令, 如组合 DNA 组装所必需的, 需要科学家明确地指定每个吸气和手动分配命令。这将在工作流中创建一个主要的瓶颈, 并使脚本生成过程容易受到相同的移错误的影响, 就像手动执行程序集一样。这就需要一个软件工具, 可以自动化这一过程的所有部分, 从设计设备库, 以生成移指令, 并提供给研究员的板/试剂设置所需的组装。在这项工作中, 我们利用我们的软件工具来自动设计一个小型组合 dna 设备库, 以及将试剂 (缓冲液、水、酶) 和 dna 部件 (图 1b) 混合成96单罐模块化的 dna 组装反应。使用该工具无需预先编程经验, 可伸缩且吞吐量高, 默认情况下为组合。我们表明, 在两个不同的自动液体处理平台上制备的克隆反应产生了正确的顺序验证的克隆, 其频率与手动 (95%) 的反应相当, 且时间明显减少。

<table><tbody><tr><td>&lt;strong&gt;Hardware / Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Freedom EVO 150 Liquid Handling Robot</td><td>Tecan</td><td></td><td>Custom liquid handler fitted with an 8-channel pipetting arm&lt;br/&gt; http://lifesciences.tecan.com/products/liquid_handling_and_robotics/freedom_evo</td></tr><tr><td>Freedom EVOware Standard (Version 2.4 Service Pack 2)</td><td>Tecan</td><td></td><td>Software used to control the Freedom EVO 150 liquid handler&lt;br/&gt; http://lifesciences.tecan.com/products/software/freedom_evoware</td></tr><tr><td>Echo 550</td><td>Labcyte</td><td></td><td>Acoustic Liquid Dispenser&lt;br/&gt; http://www.labcyte.com/products/liquidhandling/echo-550-liquid-handler</td></tr><tr><td>Sorvall Legend RT</td><td>Sorvall</td><td></td><td>Large benchtop swing-bucket sentrifuge</td></tr><tr><td>MasterCycler Pro</td><td>Eppendorf</td><td>950040015</td><td>Thermocycler with 96-well heat block&lt;br/&gt; https://online-shop.eppendorf.us/US-en/PCR-44553/Cyclers-44554/Mastercycler-pro-PF-5193.html</td></tr><tr><td>ECHOTHERM Chilling/Heating Dry Bath</td><td>Torrey Pines Scientific</td><td></td><td>Heating/Chilling block for EVO 150 deck&lt;br/&gt; https://www.torreypinesscientific.com/products/chilling-and-heating-dry-baths/echotherm-ric20-series-remote-controlled-chillingheating-dr</td></tr><tr><td>Tabletop Microcentrifuge 5418</td><td>Eppendorf</td><td>5418000017</td><td>Stardard 18-well microcentrifuge&lt;br/&gt; https://online-shop.eppendorf.com/OC-en/Centrifugation-44533/Centrifuges-44534/Centrifuges-5418--5418R-PF-9257.html</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Resources&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>mocloassembly.com</td><td>Lattice Automation</td><td></td><td>Web-tool for combinatorial DNA assembly&lt;br/&gt; mocloassembly.com</td></tr><tr><td>CIDAR MoClo Parts Kit</td><td>AddGene</td><td>1000000059</td><td>Kit of bacterial glycerol stocks for all DNA parts used in this study&lt;br/&gt; https://www.addgene.org/cloning/moclo/densmore/</td></tr><tr><td>CIDAR ICE Registry</td><td>CIDAR Lab</td><td></td><td>Registry of plasmid DNA maps&lt;br/&gt; https://ice.cidarlab.org/folders/8&lt;br/&gt; https://synbiohub.programmingbiology.org/public/bubdc_ice/bubdc_ice_folder_8/current</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Labware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 &amp;mu;L Conductive Tips</td><td>Tecan</td><td>30 057 818</td><td>Sterile 50 &amp;mu;L conductive tips for Tecan liquid handler&lt;br/&gt; http://lifesciences.tecan.com/products/consumables/disposable_tips/liquid_handling_disposable_tips</td></tr><tr><td>2 mL Deep 96-well Culture Plates</td><td>USA Scinetific</td><td>5678-0285</td><td>Bacterial culture plates used for culturing of large numbers of samples&lt;br/&gt; http://www.thermoscientific.com/en/product/nunc-1-3-2-0ml-deepwell-plates-shared-wall-technology.html</td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>USA Scinetific</td><td>1615-5599</td><td>Disposable microcentrifuge tubes&lt;br/&gt; http://www.usascientific.com/Seal-Rite-1.5-ml-tube-colors.aspx</td></tr><tr><td>Breathe Easier sealing membrane</td><td>Sigma-Aldrich</td><td>Z763624-100EA</td><td>Breathable sealing membrane for bacterial culture plates&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/z763624?lang=en&amp;amp;region=US</td></tr><tr><td>Full-Skirted, Low-Profile, 96-Well PCR Plates</td><td>GeneMate</td><td>T-3183-2</td><td>PCR plates used for all steps&lt;br/&gt; https://www.bioexpress.com/store/catalog/product.jsp?catalog_number=T-3183-R</td></tr><tr><td>Alluminum Sealing Foil for PCR Plates</td><td>GeneMate</td><td>T-2451-1</td><td>Alluminum seals for PCR plate storage&lt;br/&gt; https://www.bioexpress.com/store/catalog/product.jsp?catalog_number=T-2451-1</td></tr><tr><td>Polyolefin Sealing Film for PCR Plates</td><td>GeneMate</td><td>T-2450-1</td><td>Plastic seals for PCR plates during cycling&lt;br/&gt; https://www.bioexpress.com/store/catalog/product.jsp?catalog_number=T-2450-1</td></tr><tr><td>PCR Cooler</td><td>Eppendorf</td><td>22510525</td><td>96-well cold block&lt;br/&gt; https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Accessories-44520/PCR-Cooler-PF-55940.html</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GenCatch Plasmid DNA Mini-Prep Kit</td><td>Epoch Life Sciences</td><td>2160250</td><td>Plasmid DNA purification kit&lt;br/&gt; http://www.epochlifescience.com/Product/PurificationKit/dna_mini.aspx</td></tr><tr><td>T4 DNA Ligase (HC)</td><td>Promega</td><td>M1794</td><td>High concentration T4 DNA Ligase&lt;br/&gt; https://www.promega.com/products/cloning-and-dna-markers/molecular-biology-enzymes-and-reagents/t4-dna-ligase/?catNum=M1794</td></tr><tr><td>BbsI Restriction Enzyme</td><td>New England Biolabs</td><td>R0539L</td><td>BbsI enzyme at 10,000 units/ml&lt;br/&gt; https://www.neb.com/products/r0539-bbsi</td></tr><tr><td>BsaI Restriction Enzyme</td><td>New England Biolabs</td><td>R0535L</td><td>BsaI enzyme at 10,000 units/ml&lt;br/&gt; https://www.neb.com/products/r3535-bsai-hf</td></tr><tr><td>T4 DNA Ligase Buffer Pack</td><td>Promega</td><td>C1263</td><td>10x T4 DNA ligase buffer&lt;br/&gt; https://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/t4-dna-ligase/</td></tr><tr><td>Isopropyl-&amp;szlig;-D-thiogalactopyranoside (IPTG)</td><td>Zymo Research</td><td>I1001-25</td><td>0.5M IPTG Solution&lt;br/&gt; http://www.zymoresearch.com/buffers-solutions/chemicals/isopropyl-ss-d-thiogalactopyranoside-iptg</td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;szlig;-D-galactopyranoside (X-GAL)</td><td>Zymo Research</td><td>X1001-25</td><td>20 mg/ml X-GAL solution&lt;br/&gt; http://www.zymoresearch.com/buffers-solutions/chemicals/5-bromo-4-chloro-3-indolyl-ss-d-galactopyranoside-x-gal</td></tr><tr><td>Kanamycin Sulfate</td><td>Zymo Research</td><td>A1003-25</td><td>35 mg/ml Kanamycin solution&lt;br/&gt; http://www.zymoresearch.com/buffers-solutions/antibiotics/kanamycin-sulfate</td></tr><tr><td>Carbenicillin (Disodium Salt)</td><td>Fisher</td><td>BP26481</td><td>1 g Carbenicillin (Ampicillin analog)&lt;br/&gt; https://www.fishersci.com/shop/products/carbenicillin-disodium-salt-fisher-bioreagents-3/p-25005#?keyword=carbenicillin</td></tr><tr><td>SOC Broth Media</td><td>Teknova</td><td>S0225</td><td>Powder media used to make SOC broth&lt;br/&gt; http://www.teknova.com/SOC-BROTH-MEDIA-p/s0225.htm</td></tr><tr><td>LB Broth (Lennox) Media</td><td>Sigma-Aldrich</td><td>L3022-1KG</td><td>Powder media used to make LB broth&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/l3022?lang=en&amp;amp;region=US</td></tr><tr><td>LB Broth with agar (Lennox) Media</td><td>Sigma-Aldrich</td><td>L2897-1KG</td><td>LB with agar mix used for making solid media plates&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/l2897?lang=en&amp;amp;region=US</td></tr><tr><td>Alpha-Select Gold Efficiency Competent Cells</td><td>Bioline</td><td>BIO-85027</td><td>High efficiency chemically competent E. coli cells&lt;br/&gt; http://www.bioline.com/us/alpha-select-gold-efficiency.html</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Primer VF</td><td></td><td></td><td>5&#039;-TGCCACCTGACGTCTAAGAA-3&#039;&lt;br/&gt; Primers used for Sanger sequencing and colony PCRs</td></tr><tr><td>Primer VR</td><td></td><td></td><td>5&#039;-ATTACCGCCTTTGAGTGAGC-3&#039;&lt;br/&gt; Primers used for Sanger sequencing and colony PCRs</td></tr></tbody></table>

automated robotic liquid handling assembly of modular dna devices

在模块化 DNA 组装技术方面的最新进展使合成生物学家能够更有效地测试更多可用的 "设计空间", 这些 "装置" 是由单个基因组分的组合所构成的。但是, 手动组装如此多的设备是时间密集型的, 容易出错, 而且成本高昂。合成生物学研究的日益复杂和规模化, 需要一种高效、可重现的方法来适应大规模、复杂和高吞吐量的设备建设。
在这里, 使用基于类型 IIS 限制切的模块化克隆 (MoClo) 技术的 DNA 组装协议是在两个液体处理机器人平台上自动进行的。自动化的液体处理机器人需要仔细, 经常单调乏味的优化移参数的液体不同的粘度 (如酶, DNA, 水, 缓冲), 以及明确的编程, 以确保正确的愿望和配药的 DNA 部分和试剂。这使得手动脚本编写复杂的程序集就像手动 DNA 汇编一样成问题, 并且需要一个能够自动生成脚本的软件工具。为此, 我们开发了一个基于 web 的软件工具, http://mocloassembly.com, 用于从作为基因库文件上传的基本 dna 部件生成组合 dna 设备库。我们提供的工具的访问, 并从我们的液体处理软件的出口文件, 其中包括优化液体类, 实验室参数, 和甲板布局。所有使用的 DNA 部件都可通过 Addgene, 他们的数字地图可以通过波士顿大学的 BDC 冰上登记。这些元素共同为其他组织自动化模块化克隆实验和类似协议提供了基础。
在这里提出的自动化的 dna 组装工作流程能够实现可重复的、自动化的、高通量的 dna 设备生产, 并减少了重复人工移引起的人为错误的风险。测序数据显示, 从这个工作流程产生的自动的 DNA 组装反应是〜95% 正确的, 需要尽可能少的4% 手的时间, 比手动反应准备。

在这里, 我们展示了 96 dna 设备的自动化模块化组装的各种基本 dna 部件 (图 1b) 使用两个自动化的机器人液体处理平台。每个转录单元是一个线性排列的启动子, 核糖体结合位点, 基因和转录终止, 克隆成一个特定的目标载体。土族是许多等级遗传电路设计的一个关键组件28,29,30 , 因此是这种方法的概念的自然证明。从开始到结束, 可以在5天内实现序列验证的克隆, 并在图 1a中介绍了所提供的工作流的概览。
使用所描述的工具和协议, 我们捕获了几个指标, 在确定最佳的装配平台, 给定的克隆反应的定义数量由科学家产生。图 2a说明了在所有三模式下的反应装配时间的比较。执行时间是完成所有移步骤所需的全部时间来组装96反应, 其中包括分配所有的 DNA 部分和试剂。手动时间指的是人工参与克隆反应准备的总时间, 或运行液体处理程序的软件的设置。图 2b比较不同方法之间的成本。所提出的费用是由每种方法准备的单一反应, 包括酶的价格和一次性吸管提示, 考虑到最低的典型反应量 (20 µL 为液体处理, 10 µL 为手动, 250 nL 为声学分配器)。单克隆从所有96反应为手动和液体处理器准备的集合被测序了, 与一个子集12测序为音响分配器反应 (图 2c)。正确的序列得到了95% 的96手动和液体处理装置。83% 的声学分配器样本子集是正确的, 然而, 最后两个反应未能产生任何白色的殖民地, 可能是由于在分配 DNA 和试剂完成后, 液滴的混合不足。图 2d说明了克隆反应效率, 由白蚁群与菌落总数的比值来衡量, 这是人工 (94%) 和液体处理程序组装 (84%) 反应之间的可比性。有趣的是, 当用声学分配器缩小最后的反应体积时, 我们注意到反应效率有明显的下降, 反应是在小于1µL 的体积 (补充图 1 & 补充表 2) 中进行的。这可能是由于蒸发反应冷热, 这可能导致盐浓度增加的反应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54703/54703fig1.jpg"> 图1。自动化 DNA 设备库程序集工作流。 (a)本工作中介绍的实验工作流概述。(1) 研究人员首先上传基因库文件的所有 DNA 部件 & 目的地载体, 他们希望使用。(2) 下一步, 将包括在组件中的 DNA 部分被选定。(3) 该工具随后将生成自动液体处理程序的选择列表, 以及用于协助手动填充 DNA 部件和酶 mastermix 的平板地图。(4) 使用 DNA & 试剂板, 以及生成的选择列表, 研究人员在液体处理器上执行克隆反应的组装。(5) 一旦完成, 反应将被转换和镀为下游分析。(b)此工作中使用的 DNA 部件的列表。共三发起人, 三核糖体结合位点, 四编码序列, 和一个转录终结者使用产生的组合库96土族。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54703/54703fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54703/54703fig2.jpg"> 图2。比较三不同的反应装配方式。 (a)反应设置时间为96反应手动组装, 通过液体处理, 并由一个声学分配器。手工装配需要2ĥ10分钟, 所有这些都是准时的。液体处理程序采取了类似的时间 (2 小时6分钟) 执行移命令, 但只有一小部分的时间 (5 分钟) 是手。吸音器在执行液体传输 (5 分钟) 时花费的时间要少得多, 并采取了最少的动手时间 (5 分钟)。每个程序集方法的每个克隆反应的(b)价格。这个价格包括使用的酶的成本, 以及吸管技巧。(c)排序结果来自所有96集合的土族的单一殖民地。正确克隆的百分比在所有程序集方法中都是可比较的。注意, 只有一个子集 (12) 的完整的96组件是从声学分配器准备的样本转换。两个反应未能产生任何白色的殖民地, 可能是由于 DNA & 酶 mastermix 液滴在 OutputPlate 的混合不足。(d)对手动和液体处理剂制备反应的克隆反应效率的比较。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54703/54703fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图1。反应效率随着反应量的降低而降低。 当将反应量降低到1µL 以下时, 反应效率明显下降。这一趋势被认为有两种不同的最终 DNA 浓度;然而, 如果能容忍较低的效率, 科学家可能会选择使用较小的体积来节省试剂成本。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54703/Supplemental_Figure_1.pdf">请单击此处下载此文件.</a>
补充表 1。 表的 DNA 和酶液类参数的各种移卷。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54703/Supplemental_Table_1_V2.xlsx">请单击此处下载此文件.</a>
补充表 2。 反应效率计算。原始的 CFU 数字被给 12 96 被转换的反应手工准备和通过液体处理者。还提供了 CFU 数字, 用于测试声学分配器上的较小的最终反应量。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54703/Supplemental_Table_2.xlsx">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Automated Robotic Liquid Handling Assembly of Modular DNA Devices at JoVE.com

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

在这里, 一个自动化的工作流程, 执行模块化的 dna "设备" 组装使用的模块化克隆 dna 组装方法的液体处理机器人提出。该协议使用了一种直观的软件工具, 用于生成组合 DNA 设备库生成的液体处理程序列表, 我们使用两个液体处理平台进行演示。

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

automated-robotic-liquid-handling-assembly-of-modular-dna-devices

Research

JoVE Journal

Bioengineering

12.3K 次观看.  Boston University. 在这里, 一个自动化的工作流程, 执行模块化的 dna "设备" 组装使用的模块化克隆 dna 组装方法的液体处理机器人提出。该协议使用了一种直观的软件工具, 用于生成组合 DNA 设备库生成的液体处理程序列表, 我们使用两个液体处理平台进行演示。

视频: Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

折叠的DNA折纸一个生物反应机器人与表征

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

科研

化学

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

复杂的二维形状的从单链DNA的瓷砖自组装

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

生物化学

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

生物工程

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

遗传学

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

使用 CRISPRmass 的高通量 UAS-cDNA/ORF 质粒文库构建

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5&#39; end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.&#160;

作者聚焦:使用 CRISPRmass 简化全基因组质粒文库构建

We present a protocol for CRISPR-based modular assembly (CRISPRmass), a method for high-throughput construction of UAS-cDNA/ORF plasmid library in Drosophila using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various plasmid libraries.

模块化 DNA 设备的自动机器人液体处理组件

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折叠的DNA折纸一个生物反应机器人与表征

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一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

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荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建