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

איורים 1, 2, 3, 4, 6, 8 ולוחות 2, 3, 10, 15, 16 הם שונה מאסמכתא 29. עבודה זו נתמכה על ידי הקרן הלאומית למדע (NSF-להעניק CCF 1,117,143 וNSF-CCF 1,162,141 לGS). י &#39;-JC נתמכה על ידי מלגות ממשלה הטיוואנית. SDR נתמכה על ידי התכנית הלאומית למדע קרן בוגר מחקר המלגה (GRFP).

<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.

<html> מאמר זה מתאר שיטה להפקת שערי ndsDNA מפלסמיד דנ&quot;א הטהור ביותר. יתר על כן, פרוטוקול מוצג לאפיון ביצועי שער באמצעות assay קינטיקה הקרינה. נתוני ניסוי מראה כי מערכת הנגזר פלסמיד בביצועיו עמיתו הסינתטי שלה, גם אם המערכת סינתטית מורכבת מחוטים מטוהרים באמצעות ג&#39;ל אלקטרופורזה polyacrylamide (עמוד). סביר להניח, הביצועים המשופרים של שערים הנגזר פלסמיד הוא בעיקר בשל גבוה מאוד טוהר של ה- DNA הביולוגי. DNA סינטטי מכיל מגוון של טעויות, במחיקות מסוימות שתגרומנה oligonucleotides של n-1 אורך, וצד מוצרים אלה בדרך כלל לא הוסרו לחלוטין בעמוד או הליכי טיהור כרומטוגרפיה נוזלית ביצועים גבוהים (HPLC). שיפורים דומים לאלה שדווחו כאן גם נצפו במחקר קודם של מגבר סיכת ראש זרז שהשתמש בדנ&quot;א המופק ממקורות ביולוגיים 21. התחת = &quot;jove_content&quot;&gt; עם זאת, גם השימוש בשערים הנגזר פלסמיד לא יכולים לחסל לחלוטין טעויות בביצועי השער, שיש לפחות שתי סיבות: ראשונה על מערכת עיכול או חוסר הדיוק לחתוך יכול להוביל לשערים עם יותר מדי ניקס או חתכים בעמדות הלא נכונות. בכל מקרה, שערים נוטים יותר להשתתף בתגובות לא רצויות. בעיות מסוג זה עשויים להיות הקלה על ידי אופטימיזציה של כמות האנזים המשמשת (ראה איור 4). שנית, בניסויים אלה, רוב התשומות וגדילי דנ&quot;א סינטטי עזר היו וכך הכילו מחיקות ומוטציות. באופן עקרוני, יכולים להיות גם השיגו את כל הקלט חד-גדילים וגדילי עזר מ- DNA phagemid דרך עיכול אנזים nicking של הגנום הנגיפי M13 קידוד מראש 26. אולי ביצועי המעגל ניתן לשפר עוד יותר באמצעות ssDNA נגזר מהגנום של החיידקים. בעוד שהשימוש בשערים הנגזר פלסמיד נמצא כדי לשפר את ביצועי מעגל, ניתוחשל הפעמים העלות ועיבוד גילה כי בעוד שהייצור של שערים הנגזר פלסמיד הוא מעט זול יותר (לוח 15), זה לוקח 2-3 פעמים יותר זמן עיבוד בהשוואה להרכבה וטיהור של שערים מoligos המסונתזת מסחרי (לוח 16). העלויות העיקריות של שערים הנגזר פלסמיד הן סינתזת גן והשימוש באנזימי הגבלה. עבור 300 pmole של שערים (15 תגובות מספיק ליום 30 בננומטר), העלות המשוערת להצטרפות שערים היא כ 170 $ ו 200 $ עבור מזלג שערים, להיות הבדל העלות בשל השימוש באנזימים שסחבו שונים. לעומת זאת, הסינתזה הכימית של הגדילים לאותו שער העלויות כ 260 $ כוללים דמי טיהור עמוד. עלות הזמן העיקרית לשערים הנגזר פלסמיד היא בתהליך השכפול, ש, בדיוק כמו סינתזת DNA, יכול להיות במיקור חוץ לחברת סינתזת גן. עם זאת, יש לי שערים לאחר הרכבה, הנגזר פלסמיד היתרון שיכולים בקלות להיות משוכפלים פלסמידים המארחניתן לאחסן nd בצורה של מניות גליצרול בקטריאלי. זה מאפשר לעשות שימוש חוזר בשערים פעמים רבות. במבט קדימה, ביצועים המשופרים של שערים הנגזר פלסמיד יכולים לאפשר מגוון הרבה יותר גדול של התנהגויות דינמיקה מאשר הודגם בניסוי עד כה עם ה- DNA CRNs. לדוגמא, 47,48 עבודה תיאורטית האחרון הציע שדפוסים מרחביים באמצעות התארגנות עצמית במקרו בקנה המידה יכולים להתממש עם CRNs DNA באמצעות מנגנון דיפוזיה תגובה. השיטה המוצגת כאן מספקת נתיב קיימא לבניית הרכיבים המולקולריים שבבסיס לחומרי DNA דפוסים עצמיים כזה. למרות מאתגר, פיתוח מורפולוגיות מאקרו בקנה מידה בדרך לתכנות יהיו השלכות משמעותיות בתחומים החל מחקר בביו-חומרים לרפואת רגנרטיבית.

יכולת החיזוי של זיווג הבסיס ווטסון-קריק אפשרה DNA ננוטכנולוגיה הדינמי לצוץ כדרך הניתנת לתכנות לעיצוב התקנים מולקולריים עם תכונות דינמיות 1,2. בפרט, עקירת גדיל DNA - תגובת הכלאת תכנות, תחרותית - הוכיחה את עצמו מנגנון רב עוצמה להנדסת מערכות ה- DNA דינמיות. בתגובת עקירת גדיל DNA, oligonucleotide הנכנס דוחק גדיל קשור בעבר &quot;פלט&quot; משותף מחייב משלים. ניתן לקשר תגובות כגון מרובות יחד לתוך מפלי תגובה רב-שלבים עם רמה גבוהה של שליטה על הסדר והעיתוי של תגובה בודדת שלבים 3. מפלי עקירת גדיל DNA נעשו שימוש כדי ליצור מעגלים דיגיטליים ואנלוגיים מולקולריים 4-7, ננו החלפת 8-10, מנועים אוטונומיים מולקולריים 11-15, ומגברי קטליטי שאינם קוולנטיים 13,16-21. יתר על כן, Dניתן לדמות מכשירי NA באמצעות תגובות עקירת גדיל ומיועדים ליישומים מגוונים באמצעות תוכנת עיצוב בעזרת מחשב 22-24. נכון לעכשיו, ה- DNA מסונתז כימי משמש כחומר העיקרי לננוטכנולוגיה DNA. עם זאת, טעויות בתהליך הסינתזה של DNA, וכתוצאה מכך oligonucleotides מושלם, הם האמינו להגביל את הביצועים של מכשירי ה- DNA דינמיים על ידי גרימת תופעות תגובות שגויות. לדוגמא, תגובות &quot;דליפה&quot; יכולות לגרום לשחרורו של oligonucleotide תפוקה גם בהיעדרו של הדק תגובה. תופעות כאלה הן ברורות ביותר במפלי תגובה עצמן שבו אפילו כמות מזערית של דליפה הראשונית סופו של דבר לגרום להפעלה המלאה של 19,20 המפל. לעומת זאת, תגובות לעתים קרובות אינן מצליחות להגיע לרמה הצפויה של הפעלה כי חלק מרכיבים לא לעורר אפילו בנוכחות של הקלט המיועד 7,25. מבוסס DNA כדי להפוך את הביצועים שלננו-התקנים דומים לעמיתיהם המבוססים על החלבונים הביולוגיים שלהם, מצבי שגיאה כזו צריך להיות מופחת באופן דרמטי. פלסמידים חיידקים או DNA הביולוגי אחר יכולים לשמש כמקור זול יחסית של ה- DNA הטהור ביותר עבור יישומי ננוטכנולוגיה. כמויות גדולות של DNA יכולות להיות שנוצרו על ידי שכפול בחיידקים ויכולות ההגהה הפנימיות של מערכות חיות להבטיח את הטוהר של ה- DNA וכתוצאה מכך. למעשה, כמה מאמרים האחרונים הכירו בשירות הפוטנציאלי של ה- DNA הביולוגי ליישומי ננוטכנולוגיה 21,26-28. עם זאת, הטבע-גדילים כפולים המלאה של פלסמיד דנ&quot;א עד כה אסור שימוש בו כחומר לייצור מכשירי ה- DNA דינמיים, אשר בדרך כלל מורכב מהמספר הרב של oligonucleotides ומכילים שני תחומים פעמים התקועות וחד-גדילים. במאמר האחרון 29 סוגיה זו הייתה ממוענת ואדריכלות שער DNA חדשה שמורכבת בעיקר מגדילי דנ&quot;א כפול חתוכות (ndsDNA) היה להציגד. חשוב לציין, יכולות להיות מתוכננות מערכות שערי ndsDNA שמבינות את הדינמיקה שצוינה על ידי כל רשת פורמאלית תגובה הכימית (CRN) 29. יכולים אפוא לשמש שערי ndsDNA, בעיקרון, כדי ליצור מערכות דינמיות שמפגינות תנודות ותוהו ובוהו, bistability וזיכרון, לוגיקה בוליאנית או התנהגויות אלגוריתמיות 30-38. לדוגמא, אסמכתא. 29 הפגינו CRN שלוש-תגובה שסיפק יישום מולקולרי של פרוטוקול &quot;קונסנסוס&quot;, סוג של אלגוריתם מחשוב מבוזר 29,39,40. עבודה זו הוצגה לראשונה שימוש חדשני לפורמליזם CRN כ&quot; שפת תכנות &quot;למהירות סינתזת מערכות מולקולריות פונקציונליות (איור 1 א). כאן, פרוטוקול מפורט לנובעים שערי ndsDNA מפלסמיד דנ&quot;א מסופק. הראשון הוא סקירה של תהליך עיצוב הרצף. לאחר מכן, בעקבות הסבר של oligonucleotides איך סינטטי המכילרצפי השער הם משובטים לתוך פלסמידים והרצף אומת ומוגבר באמצעות תרבות חיידקים. לאחר מכן, הוא הראה כיצד ניתן לגזור ndsDNA שערים מפלסמידים על ידי עיבוד האנזימטית (ראה איור 2). לבסוף, שיטה לבדיקת שער התנהגות באמצעות מבחני קינטיקה הקרינה מתוארת. מנגנון תגובה כדוגמא, הפרוטוקול מתמקד בתגובה כימית + B-&gt; B + C קטליטי. מיני A, B, ו- C (&quot;אותות&quot;, איור 1) מתאים לכל מולקולת DNA חד-גדילים שונה. הרצפים של מולקולות אלה הם עצמאיים לחלוטין והגדילים לא מגיבים אחד עם השני באופן ישיר. יש הרצפים של כל האותות שני תחומים שונים פונקציונליים, כלומר, subsequences שפועל יחד בתגובות עקירת גדיל: 1) תחום דריסת רגל קצר (תוויות ת&quot;א, שחפת, TC) המשמש לייזום של r עקירת הגדילeaction, ו -2) תחום ארוך (תוויות, B, C) הקובע את זהות האות. אינטראקציות בין גדילי אות מתווכות על ידי מתחמי חתוכות פעמיים גדילי דנ&quot;א (ndsDNA) שער (נקראים הצטרפו AB ומזלג לפני הספירה) ומינים חד-גדילי עזר (&lt;tr r&gt;, &lt;b tr&gt;, &lt;ג TB&gt; ו- &lt;i TC &gt;). התגובה הרשמית + B-&gt; B + C מתבצע באמצעות סדרה של צעדי תגובת עקירת גדיל, שבו כל צעד תגובה חושף דריסת רגל לתגובה הבאה (איור 1). בדוגמא זו, אותות ו- B הם בתחילה בחינם בפתרון תוך C אות קשור לשער המזלג. בסוף התגובה B ו- C הם בפתרון. באופן כללי יותר, אותות שחייבים שער אינם פעילים ואילו אותות שהם חופשיים בפתרון הם פעילים, כלומר, הם יכולים להשתתף בתגובת עקירת גדיל כקלט. במהלך זמן התגובה ואחריו באמצעות אסטרטגית כתב ניאון (איור 1 ג). בעבודה קודמת 29, זה היה הראה כי מנגנון תגובה זו מבין stoichiometry הנכון אלא גם קינטיקה של תגובת היעד לא רק.

<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.

גדיל DNA עקירת עיר רשתות תגובת השרשת כימית המופק מפלסמיד

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 Views.  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. 

Video: 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.

עיצוב רובוט ביו תגובה מ-DNA אוריגמי

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

Bioengineering

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.

Mutagenesis האתר ביתי בימוי של פלסמידים שלמים

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

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.

אוטומטית רובוטית טיפול בנוזלים הרכבה של התקנים DNA מודולרי

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.

באמצעות ביולוגיה סינתטית מהנדס תאים חיים המתממשקות חומרים לתכנות

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.

עיצוב, סינתזה של ארון אקורדיון דנ א Reconfigurable

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.

משטח פונקציונלי-הנייח של גנים באמצעות גדיל שקודמים הזחה ליתוגרפיה

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.

הרכבה מהירה של מבנים מרובי גנים באמצעות שיבוט שער הזהב מודולרי

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.

RNA פולימראז קשור DNA לתכנות תמלול במבחנה וחישוב מולקולרי

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.

מעגלים דיגיטליים גנטיים המבוססים על מערכות CRISPR-Cas וחלבונים אנטי-קריספר

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.

דור של פלסמיד וקטורים לבטא חלבונים מתויג FLAG על פי תקנות ההסדרה של יזם אדם התארכות Factor-1α שימוש גיבסון עצרת

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