Name: protocols for c-brick dna standard assembly using cpf1
Uploaded: 2017-06-15T14:00:00
Description: Watch this Scientific Journal Video about Protocols for C-Brick DNA Standard Assembly Using Cpf1 at JoVE.com

Vi takker Shanghai Tolo Biotech for deres tekniske assistance under udviklingen af ​​C-Brick-standarden. Dette arbejde blev støttet af tilskud fra det kinesiske videnskabelige videnskabs videnskabelige prioritetsprogram (stipendium nr. XDB19040200).

1. Fremstilling af crRNA <ol><li> Fremstilling af crRNA-skabeloner <ol><li> Re-suspendere individuelle oligonukleotider ( tabel 1 ) i RNase-frit vand til en koncentration på 10 μM. </li><li> Tilsæt 22,5 μL topstrengoligonukleotid (T7-F i tabel 1 ), 22,5 μl bundstrengoligonukleotid ( tabel 1 ) og 5 μl 10x glødemiddelbuffer til et 0,2 ml PCR-rør. Sørg for, at det totale volumen er 50 μL. BEMÆRK: Seks forskellige bundstreng-oligonukleot...

<ol>
	<li>Canton, B., Labno, A., Endy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+and+standardization+of+synthetic+biological+parts+and+devices.">Refinement and standardization of synthetic biological parts and devices.</a> Nat Biotechnol. 26 (7), 787-793 (2008).</li><li>Shetty, R. P., Endy, D., Knight, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+BioBrick+vectors+from+BioBrick+parts.">Engineering BioBrick vectors from BioBrick parts.</a> J Biol Eng. 2, (2008).</li><li>Knight, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Idempotent Vector Design for Standard Assembly of Biobricks. , 1-11 (2003).</li><li>Li, S. Y., Zhao, G. P., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-Brick:+A+New+Standard+for+Assembly+of+Biological+Parts+Using+Cpf1.">C-Brick: A New Standard for Assembly of Biological Parts Using Cpf1.</a> ACS Synth Biol. 5 (12), 1383-1388 (2016).</li><li>Anderson, J. 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=BglBricks:+A+flexible+standard+for+biological+part+assembly.">BglBricks: A flexible standard for biological part assembly.</a> J Biol Eng. 4 (1), (2010).</li><li>Liu, J. K., Chen, W. H., Ren, S. X., Zhao, G. P., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iBrick:+a+new+standard+for+iterative+assembly+of+biological+parts+with+homing+endonucleases.">iBrick: a new standard for iterative assembly of biological parts with homing endonucleases.</a> PLoS One. 9 (10), e110852 (2014).</li><li>Hsu, P. D., Lander, E. S., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+applications+of+CRISPR-Cas9+for+genome+engineering.">Development and applications of CRISPR-Cas9 for genome engineering.</a> Cell. 157 (6), 1262-1278 (2014).</li><li>Wright, A. V., Nunez, J. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+Applications+of+CRISPR+Systems:+Harnessing+Nature's+Toolbox+for+Genome+Engineering.">Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering.</a> Cell. 164 (1-2), 29-44 (2016).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Zetsche, B., 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=Cpf1+is+a+single+RNA-guided+endonuclease+of+a+class+2+CRISPR-Cas+system.">Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.</a> Cell. 163 (3), 759-771 (2015).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proc Natl Acad Sci U S A. 74 (12), 5463-5467 (1977).</li><li>Engler, C., Kandzia, R., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one+pot,+one+step,+precision+cloning+method.">A one pot, one step, precision cloning method.</a> PLoS One. 3 (11), e3647 (2008).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Casini, A., Storch, M., Baldwin, G. S., Ellis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bricks+and+blueprints:+methods+and+standards+for+DNA+assembly.">Bricks and blueprints: methods and standards for DNA assembly.</a> Nat Rev Mol Cell Biol. 16 (9), 568-576 (2015).</li><li>Cameron, D. E., Bashor, C. J., Collins, J. 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Denne protokol beskriver en procedure for DNA-samlingsstandarden C-Brick. Det vigtigste trin i denne protokol er lineariseringen af ​​C-Brick standardvektoren; Ufuldstændig spaltning af vektoren kunne alvorligt påvirke succeshastigheden. Selvom Cpf1 primært spalter mål-DNA-sekvenser i &quot;18-23&quot; spaltningsmønsteret, blev der også detekteret unøjagtig spaltning nær de to baser 4 , hvilket kan forårsage et lille antal mutationer efter DNA-samling. Derfor er Sanger-sekventering n...

Standardiseringen af ​​DNA-biologiske dele er vigtig for udviklingen af ​​syntetisk biologi 1 . Udviklingen af ​​en DNA-samlingsprocedure kan erstatte ad hoc- eksperimentelle designs og fjerne mange af de uventede resultater, der opstår under samlingen af ​​genetiske komponenter i større systemer. BioBrick-standarden (BBF RFC 10) var en af ​​de tidligste foreslåede DNA-monteringsstandarder. Den anvender præfiksekvensen (indeholdende EcoRI- og XbaI-skæresites) og suffiksekvensen (indeholdende SpeI- og PstI-skæresites) 2 , 3 . Fordi XbaI og SpeI har komplementære sammenhængende ender, kan BioBrick DNA-dele, der er skåret med XbaI og SpeI, sammenføjes og generere en ny BioBrick til yderligere iterativ samling. Nogle defekter er blevet identificeret ved brug af BioBrick standard 4 . For eksempel producerer den en 8-bp arMellem DNA-delene, hvilket ikke tillader konstruktion af in-fusionsproteiner. Desuden skal de fire ovennævnte typer af 6-bp restriktionssteder fjernes fra DNA-delene, hvilket er meget ubelejligt. BglBrick-standarden blev oprettet for at løse det første problem 5 . Det skaber en 6-bp &quot;GGATCT&quot; ar, der producerer Gly-Ser og giver mulighed for fusion af flere proteiner eller protein domæner. IBrick blev udviklet til at håndtere det andet problem 6 . Det bruger homing endonucleases (HEs), som genkender lange DNA-sekvenser. Da HE-genkendelsesstederne sjældent findes i naturlige DNA-sekvenser, kan iBrick-standarden anvendes til direkte konstruktion af iBrick-dele uden at ændre deres DNA-sekvenser. Imidlertid forlader iBrick-standarden en 21 bp ar mellem DNA-delene, hvilket kan være årsagen til dens upopularitet. I de senere år har de klyngede regelmæssigt adskilt korte palindromiske gentagelser (CRISPR) system har udviklet sig hurtigt 7 , 8 . Blandt de CRISPR-associerede (Cas) proteiner anvendes Cas9 endonuklease fra Streptococcus pyogenes i vid udstrækning . Det introducerer for det meste dobbeltstrengede DNA-pauser (DSB&#39;er) med stumpe ender 9 . I 2015 karakteriserede Zhang og kollegaer Cpf1 (CRISPR fra Prevotella og Francisella 1) for første gang. Det tilhører klasse 2 type V CRISPR-Cas-systemet og er en CRISRP RNA (crRNA) -guided endonuclease 10 . I modsætning til Cas9 introducerer Cpf1 en DSB med et 4 eller 5 nt 5 &#39;overhæng 10 . Baseret på denne karakteristik blev Cpf1 brugt til at udvikle en DNA-samlingsstandard, C-Brick 4 . På en C-Brick-standardvektor flankerer fire Cpf1-målsteder af præfikset T1 / T2 og suffixet T3 / T4 de biologiske dele; Dette ligner BioBrick-standarden. Som spaltningen af ​​T2 og T3 sites pRoduces komplementære klæbrige ender, er det muligt at udføre den iterative samling af DNA-dele under dannelse af et &quot;GGATCC&quot; ar mellem delene. C-Brick-standarden har især to hovedfordele: At genkende lange målsekvenser og forlade korte ar. Den 6 bp &quot;GGATCC&quot; ar, der er genereret af C-Brick, koder for Gly-Ser, som muliggør konstruktion af fusionsproteiner. Desuden er C-Brick-standarden også delvist kompatibel med BglBrick og BioBrick standarderne.

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protocols for c-brick dna standard assembly using cpf1

CRISPR-associeret protein Cpf1 spalter dobbeltstrenget DNA under vejledning af CRISPR RNA (crRNA), der genererer klæbrige ender. På grund af denne egenskab er Cpf1 blevet anvendt til etablering af en DNA-samlingsstandard kaldet C-Brick, som har fordelen ved lange genkendelsessteder og korte ar. På en standard C-Brick-vektor er der fire Cpf1-genkendelsessteder - præfiks (T1 og T2-steder) og suffikset (T3 og T4-steder) - flankerende biologiske DNA-dele. Klyvningen af ​​T2- og T3-steder producerer komplementære klæbrige ender, hvilket muliggør samling af DNA-dele med T2- og T3-steder. I mellemtiden genereres et kort &quot;GGATCC&quot; ar mellem dele efter samling. Da det nydannede plasmid igen indeholder de fire Cpfl-spaltningssteder, muliggør fremgangsmåden den iterative samling af DNA-dele, hvilket svarer til de af BioBrick og BglBrick-standarder. En procedure, der beskriver brugen af ​​C-Brick-standarden for at samle DNA-deleEr beskrevet her. C-Brick-standarden kan i vid udstrækning anvendes af forskere, kandidater og bachelorstuderende, og endda amatører.

<html> Denne protokol demonstrerede samlingen af ​​tre kromoproteinkassetter (cjBlue (BBa_K592011), eforRed (BBa_K592012) og amilGFP (BBa_K592010)). For det første klones de kodende sekvenser af de tre ovennævnte gener og terminatorer individuelt i en C-Brick-standardvektor. Korte DNA-dele, promotor og terminator blev introduceret i C-Brick-vektoren gennem PCR-amplifikation under anvendelse af primere indeholdende de korte DNA-dele på 5&#39;-terminalen. Dette blev efterfulgt af sel...

Watch this Scientific Journal Video about Protocols for C-Brick DNA Standard Assembly Using Cpf1 at JoVE.com

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associeret protein Cpf1 kan styres af et specielt designet CRISPR-RNA (crRNA) til spaltning af dobbeltstrenget DNA på ønskede steder, der genererer klæbrige ender. Baseret på denne egenskab blev en DNA-samlingsstandard (C-Brick) etableret, og en protokol, der beskriver dens anvendelse, er beskrevet her.

Protokoller til C-Brick DNA Standard Assembly ved anvendelse af Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

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The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Immunfarvning for DNA ændringer: beregningsmæssige analyse af Konfokal billeder

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profilering DNA replikation Timing ved hjælp af zebrafisk som en In Vivo modelsystem.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Sjælden begivenhed registrering ved hjælp af fejl-korrigeret DNA og RNA sekventering

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Metoder til nøjagtig detektering af mitokondrie-DNA methylering

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

A Customizable Protocol for String Assembly gRNA Cloning STAgR

En tilpasselig protokol for streng forsamling gRNA kloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Vurdering af stamceller DNA integritet for kardiale celleterapi

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

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

High-gennemløb DNA plasmid multiplexing og Transfection ved hjælp af akustisk Nanodispenserings teknologi

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