Name: plasmid-derived dna strand displacement gates for implementing chemical reaction networks
Uploaded: 2015-11-25T13:00:00.000+00:00
Duration: 7 min 50 s
Description: Watch this Scientific Journal Video about Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks at JoVE.com

図1、2、3、4、6、8及び表2、3、10、15、16は、参考文献29から変更されています。この作品は、国立科学財団（NSF-GSにCCF 1117143およびNSF-CCF 1162141を付与する）によってサポートされていました。 Y.-JCは、台湾政府のフェローシップによってサポートされていました。 SDRは、国立科学財団大学院研究フェローシッププログラム（GRFP）によってサポートされていました。

<ol>
	<li>Zhang, D. Y., Seelig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+DNA+nanotechnology+using+strand-displacement+reactions.">Dynamic DNA nanotechnology using strand-displacement reactions.</a> Nat. Chem. 3, 103-113 (2011).</li><li>Krishnan, Y., Simmel, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+based+molecular+devices.">Nucleic acid based molecular devices.</a> Angew. Chem. Int. Ed. Engl. 50, 3124-3156 (2011).</li><li>Zhang, D. Y., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+DNA+strand+displacement+kinetics+using+toehold+exchange.">Control of DNA strand displacement kinetics using toehold exchange.</a> J. Am. Chem. Soc. 131, 17303-17314 (2009).</li><li>Qian, L., Winfree, E., Bruck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+network+computation+with+DNA+strand+displacement+cascades.">Neural network computation with DNA strand displacement cascades.</a> Nature. 475, 368-372 (2011).</li><li>Qian, L., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+up+digital+circuit+computation+with+DNA+strand+displacement+cascades.">Scaling up digital circuit computation with DNA strand displacement cascades.</a> Science. 332, 1196-1201 (2011).</li><li>Zadegan, R. M., Jepsen, M. D., Hildebrandt, L. L., Birkedal, V., Kjems, 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+fuzzy+and+boolean+logic+gates+based+on+DNA.">Construction of a fuzzy and boolean logic gates based on DNA.</a> Small. 11, 1811-1817 (2015).</li><li>Seelig, G., Soloveichik, D., Zhang, D. Y., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-free+nucleic+acid+logic+circuits.">Enzyme-free nucleic acid logic circuits.</a> Science. 314, 1585-1588 (2006).</li><li>Zadegan, R. 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=Construction+of+a+4+zeptoliters+switchable+3D+DNA+box+origami.">Construction of a 4 zeptoliters switchable 3D DNA box origami.</a> ACS Nano. 6, 10050-10053 (2012).</li><li>Andersen, E. 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=Self-assembly+of+a+nanoscale+DNA+box+with+a+controllable+lid.">Self-assembly of a nanoscale DNA box with a controllable lid.</a> Nature. 459, 73-76 (2009).</li><li>Zhang, D. Y., Hariadi, R. F., Choi, H. M., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+DNA+strand-displacement+circuitry+with+DNA+tile+self-assembly.">Integrating DNA strand-displacement circuitry with DNA tile self-assembly.</a> Nat. Commun. 4, (1965).</li><li>Yurke, B., Turberfield, A. J., Mills, A. P., Simmel, F. C., Neumann, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA-fuelled+molecular+machine+made+of+DNA.">A DNA-fuelled molecular machine made of DNA.</a> Nature. 406, 605-608 (2000).</li><li>Green, S. J., Lubrich, D., Turberfield, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+hairpins:+fuel+for+autonomous+DNA+devices.">DNA hairpins: fuel for autonomous DNA devices.</a> Biophys. J. 91, 2966-2975 (2006).</li><li>Venkataraman, S., Dirks, R. M., Rothemund, P. W., Winfree, E., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autonomous+polymerization+motor+powered+by+DNA+hybridization.">An autonomous polymerization motor powered by DNA hybridization.</a> Nat. Nanotechnol. 2, 490-494 (2007).</li><li>Green, S. J., Bath, J., Turberfield, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+chemomechanical+cycles:+a+mechanism+for+autonomous+molecular+motion.">Coordinated chemomechanical cycles: a mechanism for autonomous molecular motion.</a> Phys. Rev. Lett. 101, 238101 (2008).</li><li>Omabegho, T., Sha, R., Seeman, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bipedal+DNA+Brownian+motor+with+coordinated+legs.">A bipedal DNA Brownian motor with coordinated legs.</a> Science. 324, 67-71 (2009).</li><li>Turberfield, A. 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=DNA+fuel+for+free-running+nanomachines.">DNA fuel for free-running nanomachines.</a> Phys. Rev. Lett. 90, 118102 (2003).</li><li>Dirks, R. M., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggered+amplification+by+hybridization+chain+reaction.">Triggered amplification by hybridization chain reaction.</a> Proc. Natl. Acad. Sci. U. S. A. 101, 15275-15278 (2004).</li><li>Seelig, G., Yurke, B., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalyzed+relaxation+of+a+metastable+DNA+fuel.">Catalyzed relaxation of a metastable DNA fuel.</a> J. Am. Chem. Soc. 128, 12211-12220 (2006).</li><li>Zhang, D. Y., Turberfield, A. J., Yurke, B., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+entropy-driven+reactions+and+networks+catalyzed+by+DNA.">Engineering entropy-driven reactions and networks catalyzed by DNA.</a> Science. 318, 1121-1125 (2007).</li><li>Yin, P., Choi, H. M., Calvert, C. R., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+biomolecular+self-assembly+pathways.">Programming biomolecular self-assembly pathways.</a> Nature. 451, 318-322 (2008).</li><li>Chen, X., Briggs, N., McLain, J. R., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stacking+nonenzymatic+circuits+for+high+signal+gain.">Stacking nonenzymatic circuits for high signal gain.</a> Proc. Natl. Acad. Sci. U. S. A. 110, 5386-5391 (2013).</li><li>Phillips, A., Cardelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programming+language+for+composable+DNA+circuits.">A programming language for composable DNA circuits.</a> J. R. Soc. Interface. 6, S419-S436 (2009).</li><li>Lakin, M. R., Youssef, S., Polo, F., Emmott, S., Phillips, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+DSD:+a+design+and+analysis+tool+for+DNA+strand+displacement+systems.">Visual DSD: a design and analysis tool for DNA strand displacement systems.</a> Bioinformatics. 27, 3211-3213 (2011).</li><li>Lakin, M. R., Youssef, S., Cardelli, L., Phillips, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abstractions+for+DNA+circuit+design.">Abstractions for DNA circuit design.</a> J. R. Soc. Interface. 9, 470-486 (2012).</li><li>Zhang, D. Y., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robustness+and+modularity+properties+of+a+non-covalent+DNA+catalytic+reaction.">Robustness and modularity properties of a non-covalent DNA catalytic reaction.</a> Nucleic Acids Res. 38, 4182-4197 (2010).</li><li>Ducani, C., Kaul, C., Moche, M., Shih, W. M., Hogberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+production+of+'monoclonal+stoichiometric'+single-stranded+DNA+oligonucleotides.">Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.</a> Nat. Methods. 10, 647-652 (2013).</li><li>Lin, 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=In+vivo+cloning+of+artificial+DNA+nanostructures.">In vivo cloning of artificial DNA nanostructures.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 17626-17631 (2008).</li><li>Bhatia, D., 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=Icosahedral+DNA+nanocapsules+by+modular+assembly.">Icosahedral DNA nanocapsules by modular assembly.</a> Angew. Chem. Int. Ed. Engl. 48, 4134-4137 (2009).</li><li>Chen, Y. 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=Programmable+chemical+controllers+made+from+DNA.">Programmable chemical controllers made from DNA.</a> Nat. Nanotechnol. 8, 755-762 (2013).</li><li>Arkin, A., Ross, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+functions+in+biochemical+reaction+networks.">Computational functions in biochemical reaction networks.</a> Biophys. J. 67, 560-578 (1994).</li><li>&#201;rdi, P., T&#243;th, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mathematical models of chemical reactions: theory and applications of deterministic and stochastic models. , (1989).</li><li>Magnasco, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+kinetics+is+Turing+universal.">Chemical kinetics is Turing universal.</a> Phys. Rev. Lett. 78, 1190 (1997).</li><li>Oishi, K., Klavins, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+implementation+of+linear+I/O+systems.">Biomolecular implementation of linear I/O systems.</a> IET Syst. Biol. 5, 252-260 (2011).</li><li>Senum, P., Riedel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate-independent+constructs+for+chemical+computation.">Rate-independent constructs for chemical computation.</a> PLoS One. 6, (2011).</li><li>Soloveichik, D., Cook, M., Winfree, E., Bruck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computation+with+finite+stochastic+chemical+reaction+networks.">Computation with finite stochastic chemical reaction networks.</a> Natural Computing. 7, 615-633 (2008).</li><li>Soloveichik, D., Seelig, G., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+as+a+universal+substrate+for+chemical+kinetics.">DNA as a universal substrate for chemical kinetics.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 5393-5398 (2010).</li><li>Tyson, J. J., Chen, K. C., Novak, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sniffers,+buzzers,+toggles+and+blinkers:+dynamics+of+regulatory+and+signaling+pathways+in+the+cell.">Sniffers, buzzers, toggles and blinkers: dynamics of regulatory and signaling pathways in the cell.</a> Curr. Opin. Cell. Biol. 15, 221-231 (2003).</li><li>Cardelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-domain+DNA+strand+displacement.">Two-domain DNA strand displacement.</a> Math. Struct. Comput. Sci. 23, 247-271 (2013).</li><li>Angluin, D., Aspnes, J., Eisenstat, 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+simple+population+protocol+for+fast+robust+approximate+majority.">A simple population protocol for fast robust approximate majority.</a> Distrib. Comput. 21, 87-102 (2008).</li><li>Cardelli, L., Csikasz-Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+cycle+switch+computes+approximate+majority.">The cell cycle switch computes approximate majority.</a> Sci. Rep. 2, 656 (2012).</li><li>Zadeh, J. N., 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=NUPACK:+Analysis+and+design+of+nucleic+acid+systems.">NUPACK: Analysis and design of nucleic acid systems.</a> J. Comput. Chem. 32, 170-173 (2011).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J. Vis. Exp. , (2012).</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, 343-345 (2009).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+plasmid+DNA+into+E.+coli+using+the+heat+shock+method.">Transformation of plasmid DNA into E. coli using the heat shock method.</a> J. Vis. Exp. , e253 (2007).</li><li>Lessard, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+E.+coli+via+electroporation.">Transformation of E. coli via electroporation.</a> Methods Enzymol. 529, 321-327 (2013).</li><li>Nasri, M., Thomas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+the+specificity+of+PvuII+restriction+endonuclease.">Alteration of the specificity of PvuII restriction endonuclease.</a> Nucleic Acids Res. 15, 7677-7687 (1987).</li><li>Dalchau, N., Seelig, G., Phillips, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+design+of+reaction-diffusion+patterns+using+DNA-based+chemical+reaction+networks.">Computational design of reaction-diffusion patterns using DNA-based chemical reaction networks.</a> DNA Computing and Molecular Programming. , 84-99 (2014).</li><li>Scalise, D., Schulman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+modular+reaction-diffusion+programs+for+complex+pattern+formation.">Designing modular reaction-diffusion programs for complex pattern formation.</a> Technology. 2, 55-66 (2014).</li></ol>

The authors declare no competing financial interests.

本論文では、高純度のプラスミドDNAからndsDNAゲートを導出するための方法を説明します。また、プロトコルは、蛍光動力学アッセイを用いて、ゲート性能を特徴付けるために提示されています。実験データは、プラスミド由来の系、合成系は、ポリアクリルアミドゲル電気泳動（PAGE）を用いて精製したストランドから組み立てられていても、その合成対応物よりも優れていることを示しています。多分、プラスミド由来のゲートの性能の改善は、主に生物学的DNAの非常に高純度に起因するものです。合成DNAは、さまざまなエラーが含まれ、特に、長さN-1のオリゴヌクレオチド、およびそのような副生成物を生じる欠失は、典型的には、完全PAGEまたは高速液体クロマトグラフィー（HPLC）精製手順で除去されません。ここで報告されたものと同様の改善はまた、生物学的供給源21に由来するDNAを使用した触媒ヘアピン増幅器の以前の研究で観察されました。 <p clお尻= &quot;jove_content&quot;&gt;しかし、プラスミド由来のゲートを用いても、完全に少なくとも2つの理由がありますするためのゲート性能のエラー、排除することはできません。あまりにも多くてゲートにつながることができ、最初の過消化またはカット精度の欠如を間違った位置にニックまたはニック。いずれの場合においても、ゲートが望ましくない反応に関与する可能性が高いです。このような問題は、（ 図4を参照）を使用する酵素の量を最適化することにより緩和することができます。第二に、これらの実験では、ほとんどの入力および補助鎖合成DNAであり、したがって、欠失および変異を含みました。原則として、すべての一本鎖入力と補助鎖はまた、事前符号化M13ウイルスゲノム26のニッキング酵素消化を介してファージミドDNAから得ることができます。おそらく回路性能は、さらに、細菌ゲノムに由来​​する一本鎖DNAを用いて改善することができます。 プラスミド由来のゲートの使用は、回路性能を改善することが見出されたが、分析コストや処理時間のプラスミド由来のゲートの製造がやや安い（ 表15）であるが、それは商業的に合成されたオリゴ（ 表16）からゲートのアセンブリおよび精製に比べて2~3倍長い処理時間を要することが明らかになりました。プラスミド由来のゲートの主要コストは、遺伝子合成、制限酵素の使用です。 （30 nmで15の反応のために十分な）ゲートの300ピコモルのために、参加ゲートの推定コストはフォークゲート、違いによるニッキング酵素の使用にいるコストの差は約$ 170と$ 200です。これとは対照的に、PAGE精製料込み$ 260の周りに同じゲー​​ト費用鎖の化学合成。プラスミド由来のゲートのための主要な時間コストは、単にDNA合成と同様に、遺伝子合成会社に委託することができ、クローニング手順、です。しかし、一旦組み立て、プラスミド由来のゲートは、宿主プラスミドを容易に複製することができるという利点を有しますND細菌のグリセロールストックの形で保存することができます。これは、上にゲートを何度も再利用することができます。 楽しみにして、プラスミド由来のゲートの改良された性能を実験的にDNA CRNsで、これまでに実証されているよりも、力学挙動のはるかに大きな範囲を可能にすることができます。例えば、最近の理論的研究47,48は、マクロスケールでの自己組織化の空間パターンは、反応拡散機構を介してDNA CRNsで実現することができることを示唆しました。ここで紹介する方法は、自己パターニングDNA材料の基礎となる分子成分を構築するための実行可能なパスを提供します。挑戦が、プログラム可能な方法で、マクロスケールの形態を開発するバイオマテリアルの研究から再生医療に至るまでの分野で重要な意味を持っているでしょう。

ワトソン-クリック塩基対の予測可能性は、動的なDNAのナノテクノロジーは、動的特性1,2と分子デバイスを設計するためのプログラム可能な方法として出現することができました。特に、DNA鎖置換 - プログラム可能な、競合ハイブリダイゼーション反応は - 動的DNAシステムを操作するための強力な機構であることが判明しました。 DNAの鎖置換反応では、入ってくるオリゴヌクレオチドは、相補的結合パートナーか​​らの以前に結合した「出力」鎖を置換します。複数のそのような反応は、個々の反応3ステップの順序やタイミングを制御度の高い多段階反応カスケードに一緒に連鎖させることができます。 DNA鎖変位カスケードは4-7、切り替え可能なナノ構造体8-10、自律分子モーター11-15、および非共有結合触媒アンプ13,16-21デジタルとアナログの分子回路を生成するために使用されています。また、D鎖置換反応を用いてNAデバイスをシミュレートし、コンピュータ支援設計ソフトウェア22-24を使用して 、多様な用途のために設計することができます。 現在、化学的に合成されたDNAは、DNAナノテクノロジーのための主材料として機能します。しかし、DNA合成プロセスにおけるエラー、そして得られた不完全なオリゴヌクレオチドは、誤った副反応を引き起こすことにより、動的DNAデバイスの性能を制限すると考えられています。例えば、「リーク」反応も反応トリガーの非存在下での出力オリゴヌクレオチドの放出をもたらすことができます。このような効果は、最初のリークであっても最小限の量は、最終的にカスケード19,20の完全な活性化につながる自己触媒反応のカスケードに最も明白です。一部のコンポーネントが意図した入力7,25の存在下でさえも誘発しないので逆に、反応はしばしば活性化の期待されるレベルに到達することができません。 DNAベースの性能を作成するにはそれらの生物学的なタンパク質ベースの対応物に匹敵するナノデバイスは、エラーモードが劇的に低減する必要があります。 細菌プラスミドまたは他の生物学的DNAは、ナノテクノロジーのアプリケーションのための高純度のDNAの比較的安価な供給源として働く可能性があります。 DNAの大量の細菌中で複製することにより生成することができ、生物系の固有の校正能力は、得られたDNAの純度を確保します。実際には、いくつかの最近の論文は、ナノテクノロジーのアプリケーション21,26-28のための生物学的DNAの潜在的な有用性を認識しています。しかしながら、プラスミドDNAの完全に二本鎖の性質は、これまで一般的に複数のオリゴヌクレオチドからなり、二本鎖および一本鎖ドメインの両方を含む動的DNAデバイスを製造するための材料としての使用を禁止しています。最近の論文29この問題が対処された、主にニックの入った二本鎖DNA（ndsDNA）で構成されて新たなDNAのゲートアーキテクチャでは紹介しましたD。 重要なことには、ndsDNAゲートのシステムは、正式な化学反応ネットワーク（CRN）29で指定されたダイナミクスを実現するように設計することができます。 ndsDNAゲートは、このように振動とカオス、双安定性とメモリ、ブール論理またはアルゴリズムの挙動30-38を示 ​​す動的システムを作成するには、原則として、使用することができます。例えば、参考文献29は、「コンセンサス」プロトコル、分散コンピューティングアルゴリズム29,39,40のタイプの分子の実装を提供三反応CRNを示しました。この作品は、最初に急速に機能性分子システムを合成するための「プログラミング言語」（ 図1A）などのCRNの形式化のための新規な使用を実証しました。 ここでは、プラスミドDNAからndsDNAゲートを導出するための詳細なプロトコルが提供されます。最初のシーケンス設計プロセスのレビューです。そして、どのように含む合成オリゴヌクレオチドの説明を次のゲート配列は、細菌培養物を介して検証され、増幅されたプラスミドとの配列中にクローニングされます。 ndsDNAゲートは、酵素処理によりプラスミドから誘導することができる方法を次に、それが示されている（ 図2参照）。最後に、蛍光動力学アッセイを用いてゲートの動作をテストするための方法を概説します。 反応機構 例として、プロトコルは、触媒化学反応A + B-&gt; B + Cに焦点を当てています。種A、B、およびC（「シグナル」、 図1B）は、すべての異なる一本鎖DNA分子に対応します。これらの分子の配列は完全に独立しており、鎖は互いに直接反応しません。すべての信号のシーケンス、すなわち二つの異なる機能的ドメイン、鎖置換反応において一緒に作用する部分配列を有する：1）短い足掛かりドメイン（ラベル鎖置換rの開始のために使用され、TB、TC）TA信号の同一性を決定するeaction、及び2）長いドメイン（A、B、Cのラベル）。 信号鎖間の相互作用は、ニックの入った二本鎖DNA（ndsDNA）ゲート複合体（ABとフォークBCに参加呼ばれる）と補助一本鎖種（&lt;TR R&gt;、&lt;BのTR&gt;、&lt;CのTB&gt;と&lt;私のTCによって媒介されます&gt;）。正式な反応A + B-&gt; B + Cは、各反応工程は、その後の反応（ 図1B）のための足掛かりを公開鎖置換反応の一連のステップを介して実行されます。信号Cは、フォークゲートに結合している間に、この例では、信号A及びBは、最初に溶液中で遊離しています。反応BとCの終了時に溶液中にあります。より一般的には、ゲートにバインドされた信号は、溶液中で遊離している信号がアクティブな間に、つまり、彼らは鎖置換反応などに参加することができ、非アクティブです入力。反応の時間経過は、蛍光レポーター戦略（ 図1C）を使用して続いています。以前の研究29には、この反応機構は、正しい化学量論だけでなく、目的とする反応の速度を実現するだけでなく、ことが示されました。

<table><tbody><tr><td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td><td>NEB</td><td>M0531S</td><td></td></tr><tr><td>PvuII-HF</td><td>NEB</td><td>R3151L</td><td></td></tr><tr><td>PstI-HF</td><td>NEB</td><td>R3140S</td><td></td></tr><tr><td>Gibson Assembly Master Mix</td><td>NEB</td><td>E2611S</td><td></td></tr><tr><td>Terrific Broth, Modified</td><td>SIGMA-ALDRICH</td><td>T0918-250G</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (250)</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>QIAGEN Hispeed Maxi-prep Kit</td><td>QIAGEN</td><td>12662</td><td></td></tr><tr><td>Nb.BsrDI</td><td>NEB</td><td>R0648L</td><td></td></tr><tr><td>Nt.BstNBI</td><td>NEB</td><td>R0607L</td><td></td></tr><tr><td>NanoDrop 2000c</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Double-stranded Genomic Blocks</td><td>IDT</td><td></td><td></td></tr><tr><td>Horiba Jobin-Yvon Spex Fluorolog-3 Fluorimeter</td><td>Horiba/Jobin Yvon</td><td></td><td></td></tr><tr><td>Synthetic Quartz Cells&amp;nbsp;</td><td>Starna</td><td>23-5.45-S0G-5</td><td></td></tr><tr><td>QIAGEN Gel Extraction Kit</td><td>QIAGEN</td><td>28706</td><td></td></tr><tr><td>Plasmid Backbones</td><td>BioBrick</td><td>E0240-pSB1A2</td><td>High copy number plasmid with Ampicillin resistance. Sequence can be found from http://parts.igem.org</td></tr></tbody></table>

plasmid-derived dna strand displacement gates for implementing chemical reaction networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Watch this Scientific Journal Video about Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks at JoVE.com

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

実装化学反応ネットワーク用プラスミド由来のDNA鎖置換ゲイツ

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

plasmid-derived-dna-strand-displacement-gates-for-implementing

Universidad San Sebastian

Research

JoVE Journal

Biology

14.3K ビュー.  University of Washington. This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language. 

ビデオ: Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

DNA折り紙からバイオ応答ロボットの設計

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

生物工学

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

全プラスミドの自家製のサイトが特異的突然変異誘発

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

生物学

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

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

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

プログラム可能な材料とのインタフェースリビング細胞を操作するために合成生物学を使用して、

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

再構成可能な DNA アコーディオン ラックの設計と合成

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

多段鎖変位リソグラフィーを用いた遺伝子の機能的な面を固定化

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

モジュラーゴールデンゲートクローニングを用いた多遺伝子構造の迅速な組み立て

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

プログラム可能なインビトロ転写と分子計算のためのDNAテザリングRNAポリメラーゼ

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

CRISPR-Casシステムと抗CRISPRタンパク質に基づく遺伝子デジタル回路

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

ギブソンアセンブリを使用して、ヒト伸長因子1αプロモーターの制御下でFLAGタグ化タンパク質を発現するプラスミドベクターの作製

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

実装化学反応ネットワーク用プラスミド由来のDNA鎖置換ゲイツ

要約

さらに動画を探す

この動画の章

DNA折り紙からバイオ応答ロボットの設計

全プラスミドの自家製のサイトが特異的突然変異誘発

モジュラー DNA デバイスの液体ハンドリングアセンブリを自動化

プログラム可能な材料とのインタフェースリビング細胞を操作するために合成生物学を使用して、

再構成可能な DNA アコーディオンラックの設計と合成

多段鎖変位リソグラフィーを用いた遺伝子の機能的な面を固定化

モジュラーゴールデンゲートクローニングを用いた多遺伝子構造の迅速な組み立て

プログラム可能なインビトロ転写と分子計算のためのDNAテザリングRNAポリメラーゼ

CRISPR-Casシステムと抗CRISPRタンパク質に基づく遺伝子デジタル回路

ギブソンアセンブリを使用して、ヒト伸長因子1αプロモーターの制御下でFLAGタグ化タンパク質を発現するプラスミドベクターの作製

実装化学反応ネットワーク用プラスミド由来のDNA鎖置換ゲイツ

要約

さらに動画を探す

この動画の章

DNA折り紙からバイオ応答ロボットの設計

全プラスミドの自家製のサイトが特異的突然変異誘発

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

プログラム可能な材料とのインタフェースリビング細胞を操作するために合成生物学を使用して、

再構成可能な DNA アコーディオン ラックの設計と合成

多段鎖変位リソグラフィーを用いた遺伝子の機能的な面を固定化

モジュラーゴールデンゲートクローニングを用いた多遺伝子構造の迅速な組み立て

プログラム可能なインビトロ転写と分子計算のためのDNAテザリングRNAポリメラーゼ

CRISPR-Casシステムと抗CRISPRタンパク質に基づく遺伝子デジタル回路

ギブソンアセンブリを使用して、ヒト伸長因子1αプロモーターの制御下でFLAGタグ化タンパク質を発現するプラスミドベクターの作製

モジュラー DNA デバイスの液体ハンドリングアセンブリを自動化

再構成可能な DNA アコーディオンラックの設計と合成