Autores gostariam de reconhecer Malcom Campbell e Todd Eckdahl com o Consórcio do Genoma para o Active Ensino (GCAT) e da Educação Alliance HHMI-Science - programa Caçadores de fagos Avançando Genómica e Ciência Evolutiva (SEA-fagos). Este projecto foi apoiado por doações do Centro Nacional de Investigação Recursos (P20RR016460) e do Instituto Nacional de Ciências Médicas Gerais (P20GM103429) do National Institutes of Health. Esta pesquisa foi apoiada em parte pela National Science Foundation, Grant # IIA-1.457.888. (Ouachita Baptist University) Os fundos Além disso institucionais foram fornecidos pela JD Patterson Verão Research Fellowship.

1. Projecção e Ordenação oligonucleótidos com as apropriadas pegajosas Termina <ol><li> Identificar potenciais terminadores independente de rho através da análise genômica usando programas que estão livremente disponíveis online 7. </li><li> Quando se trabalha com o ADN de cadeia dupla, determinar a orientação do terminador para ser testado. 7 O plasmídeo PGR-azul apenas verifica terminadores ligados na direcção 5 &#39;para 3&#39; na parte superior (vertente para a frente). <ol><li> Converter um fio terminator inferior (reverso) para o seu complemento revertida para reorientar a sequência de ensaios in PGR-azul usando software livre online, por exemplo, APE. </li></ol></li><li> Após a orientação é determinada para ser correta, adicionar a extremidade coesiva &quot;5&#39;- CGAC-3 &#39;&quot; para a extremidade 5&#39; da cadeia superior. Adicionar a extremidade coesiva &quot;5&#39;- CCGC-3 &#39;&quot; para a extremidade 5&#39; da cadeia inferior. 8 Isto irá assegurar que pastilha ligado irá ser inserido no adequadoorientação usando GGA. </li><li> Uma vez que as sequências são determinadas, pedir como oligonucleotídeos de cadeia simples comercialmente. </li></ol> 2. Annealing oligonucleotídeos (aplica apenas para liofilizadas DNA) <ol><li> Re-suspender oligonucleótidos individuais em água livre de nuclease, a uma concentração de 100 uM. </li><li> Adicione 10x tampão de emparelhamento: NaCl 1 M e Tris-HCl 100 mM pH 7,4. tampão de emparelhamento pode ser armazenado a 4 ° C durante várias semanas. Uma vez que é feita de tampão de recozimento e os oligonucleótidos são re-suspensas, iniciar o recozimento 8. </li><li> Para cada terminador a ser testado, adicionar 16 ul de água ultra-pura para um tubo de microcentrífuga de 1,5 ml. </li><li> Adicionar 2 l de 10x tampão de têmpera a cada tubo de microcentrífuga. </li><li> Adicionar 1 ml de topo oligonucleótido cadeia (100 um) a partir do terminador desejado. </li><li> Adicionar 1 ml de oligonucleótido inferior cadeia (100 um) a partir do terminador desejado. </li><li> Criar uma ebulição ba de águath utilizando um copo de 1000 ml contendo cerca de 400 ml de água da torneira e coloque em um prato quente. </li><li> Coloque os tubos de microcentrífuga a partir do passo 2.6 em uma bóia e deixe ferver por 4 min. Após 4 min, desligue o calor de placas, mas deixar o tubo e flutuar no banho de água para arrefecer lentamente O / N (18 hr) 8. Armazenar os terminadores recozido à temperatura de -20 ° C. </li></ol> 3. Montagem Golden Gate (Mix Standard - Custo Eficiente) <ol><li> Fazer uma diluição de 40 nM dos terminadores recozidos. Adicionar 124 mL de água livre de nuclease para 1 ml de mistura terminadores recozido (100 M). <ol><li> Centrifugar as amostras durante 30 segundos a 10.000 xg e armazenar no gelo. </li></ol></li><li> Em uma nova adequada do tubo para o termociclador sendo usado, adicionar 6 mL de água livre de nuclease. </li><li> Adicionar 1 mL de tampão de ligase de ADN comercial [Tris-HCl 300 (pH 7,7-7,8), MgCl2 100 mM, DTT 100 mM e 10 mM de ATP]. Execute todas as etapas subsequentes no gelo. </li><li> Adicionar1 ul de PGR-Azul (35-50 ng / ul) do plasmídeo de destino para o tubo de microcentrífuga. </li><li> Adicionar 1 mL do 40 nm terminadores emparelhado com o tubo de microcentrífuga. </li><li> Adicionar 0,5 uL de endonuclease de restrição comercial de alta fidelidade BSAI ao tubo de microcentrífuga. </li><li> Adicionar 0,5 uL de ligase de ADN comercial para o tubo de microcentrífuga. </li><li> Centrifugar a mistura reaccional a 10.000 xg, durante 120 segundos. Em seguida, coloque o tubo diretamente no termociclador. </li><li> Executar o programa de termociclador: 20 ciclos de 1 min a 37 ° C, seguido de 1 min a 16 ° C, seguido de 1 ciclo de 15 min a 37 ° C. Use amostras imediatamente ou congelar e armazenar a -20 ° C. 8 </li></ol> 4. Montagem Golden Gate (Comercial Mix Master - tempo eficiente) <ol><li> Adicionar 124 uL de água livre de nuclease e 1 uL de 100 uM terminadores criados no passo 2. Este dilui a concentração de ADN de 40 nM terminador recozidos.Execute esta etapa em uma nova, rotulados 1,5 ml tubo de microcentrífuga. </li><li> Em uma nova adequada do tubo para o termociclador, adicione 14 mL de água livre de nuclease. Verifique se o novo tubo é rotulado em conformidade. </li><li> Adicionar 2 mL de Commercial Mestre Mix Buffer para o tubo de microcentrífuga. </li><li> Adicionar 1 ml de PGR Azul plasmídeo (35-50 ng / ul) ao tubo de microcentrífuga. </li><li> Adicionar 2 mL da 40 nM terminadores emparelhado com o tubo de microcentrífuga. </li><li> Adicionar 1 ml de Commercial Master Mix ao tubo de microcentrífuga. </li><li> Centrifuga-se o tubo de 40 nm a 10000 xg durante 120 seg. Em seguida, coloque o tubo diretamente no termociclador. </li><li> Executar o programa de termociclador: 1 ciclo de 60 min a 37 ° C, seguido de 1 ciclo de 15 min a 55 ° C. Use amostras imediatamente ou congelar e armazenar a -20 ° C. </li></ol> 5. Transformação e Selecção Colony <ol><li> Prepare Luria Base (LB) agar placas de Petri contendo concentrat 1 mMion de ampicilina (Amp) e uma concentração de 10 mM de arabinose (ARB). Preparar pelo menos 10 ml de uma solução M de L da -arabinose porque esta solução de reserva vai ser usado em passos posteriores. </li><li> Use qualquer alta eficiência (10 8 - 10 9 transformantes / mg) E. quimicamente competente coli (JM109), as células para a transformação. 9, para obter os melhores resultados, adicionar 3-5 uL da reacção de ligação a 50 ul de células competentes. Quando se utiliza células competentes químicos, seguir o protocolo de transformação específica para as células a ser usadas. </li><li> Espalhe metade (25 ul) das células transformadas em LB pré-aquecido (Amp / ARB) placas de agar utilizando uma forma de L &quot;stick de hóquei&quot; estéril e incubar O / N a 37 ° C. Devido à rápida taxa de ampicilina de decomposição, não se incubar a 37 ° C ao longo de 24 h. </li><li> Realizar a seleção de colónia com base na cor por inspeção visual. A ligadura de sucesso deve ser branco / amarelo na luz visível e fluorescência verde undeazul r (450 nm) ou luz UV. Uma ligação bem sucedida irá produzir uma colônia que é de cor azul sob luz visível após 18-20 horas. </li></ol> 6. Verificação e Quantificação de Terminator <ol><li> Prepare 5 ml estéreis tubos de caldo LB com ampicilina 1 mM e 10 mM de arabinose. O número de tubos necessário é dependente do número de terminais a ser testado, mais controlos (células com resistência à ampicilina que não apresentam fluorescência e as células contendo o plasmídeo PGR-azul não cortadas). </li><li> Sob a luz visível pegar 2-3 colónias branco / amarelo (passo 5.4) usando um loop estéril. Deixar as amostras a incubar em caldos num agitador a 37 ° C e 160 rpm durante 16-18 horas. Não incubar as amostras durante mais de 24 horas. <ol><li> Opcionalmente, utilizar um espectrofotómetro para determinar a densidade celular óptica a 600 nm (DO600) para garantir o crescimento celular adequado. Geralmente, uma DO 600 de 0,8-1,0 ~ é observada após 16-18 horas de crescimento. </li></ol></li><li> Usaruma placa de 96 poços estéreis para as etapas restantes. Realizar estas etapas num ambiente asséptico e em triplicado. Adicionar 199 ul de estéril LB + Amp caldo / arabinose (1 mM de ampicilina / arabinose a 10 mM) a cada poço. Incluir quarto na placa por três controles separados. <ol><li> Use os três controlos seguintes: somente um poço (1) contendo LB + Amp / arabinose caldo e um poço (2) para células competentes não transformadas (em branco) para servir como um espaço em branco. Para a quantificação (3) da força terminador, as células que contêm PGR originais sem cortes ou contendo PGR-azul ligado com uma sequência não-terminating deve ser incluído (controlo de referência para a quantificação). Veja a Figura 1 para o layout do leitor de placas. </li></ol></li><li> Pipetar 1 mL da solução de células a partir de O / N caldos em poços individuais da placa de 96 poços. </li><li> placas de vedação com uma cobertura respirável e agitar durante 18-20 horas a 37 ° C e 160 rpm. </li><li> Utilizando um leitor de placa, determinar pela leitura OD600absorvância a 600 nm. Medir a fluorescência de GFP, como se segue: 395 de excitação e emissão de fluorescência 509. Medida RFP como se segue: 575 de excitação e de emissão 605. Após 18-20 horas, o OD 600 deve ser de aproximadamente 0,5. </li><li> Para normalizar as variações em crescimento, a utilização da equação: <img alt="equação 1" src="/files/ftp_upload/54064/54064eq1.jpg" /> tanto para GFP e RFP. </li><li> Após a normalização, determinar a força terminator relativa usando a equação <img alt="equação 2" src="/files/ftp_upload/54064/54064eq2.jpg" /></li></ol>

<ol>
	<li>Li, J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+promoter+sequence+and+its+strength+in+gene+expression.">Relationship between promoter sequence and its strength in gene expression.</a> Eur Phys J E Soft Matter. 37 (9), (2014).</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+with+high+throughput+capability.">A one pot, one step, precision cloning method with high throughput capability.</a> PLoS One. 3 (11), e3647 (2008).</li><li>Lampropoulos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GreenGate---a+novel,+versatile,+and+efficient+cloning+system+for+plant+transgenesis.">GreenGate---a novel, versatile, and efficient cloning system for plant transgenesis.</a> PLoS One. 8 (12), e83043 (2013).</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=Characterization+of+582+natural+and+synthetic+terminators+and+quantification+of+their+design+constraints.">Characterization of 582 natural and synthetic terminators and quantification of their design constraints.</a> Nat Methods. 10 (7), 659-664 (2013).</li><li>Woodin, T., Carter, V. C., Fletcher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+and+change+in+biology+undergraduate+education,+a+call+for+action--initial+responses.">Vision and change in biology undergraduate education, a call for action--initial responses.</a> CBE Life Sci Educ. 9 (2), 71-73 (2010).</li><li>Vasaly, H. L., Feser, J., Lettrich, M. D., Correa, K., Denniston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+and+change+in+the+biology+community:+snapshots+of+change.">Vision and change in the biology community: snapshots of change.</a> CBE Life Sci Educ. 13 (1), 16-20 (2014).</li><li>Naville, M., Ghuillot-Gaudeffroy, A., Marchais, A., Gautheret, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARNold:+a+web+tool+for+the+prediction+of+Rho-independent+transcription+terminators.">ARNold: a web tool for the prediction of Rho-independent transcription terminators.</a> RNA Biol. 8 (1), 11-13 (2011).</li><li>Campbell, A. 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=pClone:+Synthetic+Biology+Tool+Makes+Promoter+Research+Accessible+to+Beginning+Biology+Students.">pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students.</a> CBE Life Sci Educ. 13 (2), 285-296 (2014).</li><li>Rhee, J. I., 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=Influence+of+the+medium+composition+and+plasmid+combination+on+the+growth+of+recombinant+Escherichia+coli+JM109+and+on+the+production+of+the+fusion+protein+EcoRI::SPA.">Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA.</a> J Biotechnol. 55 (2), 69-83 (1997).</li><li>Dirla, S., Chien, J. Y., Schleif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+mutations+in+the+Escherichia+coli+AraC+protein.">Constitutive mutations in the Escherichia coli AraC protein.</a> J Bacteriol. 191 (8), 2668-2674 (2009).</li><li>Schleif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+L-arabinose+operon+of+Escherichia+coli.">Regulation of the L-arabinose operon of Escherichia coli.</a> Trends Genet. 16 (12), 559-565 (2000).</li><li>Kosuri, 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=Composability+of+regulatory+sequences+controlling+transcription+and+translation+in+Escherichia+coli.">Composability of regulatory sequences controlling transcription and translation in Escherichia coli.</a> Proc Natl Acad Sci U S A. 110 (34), 14024-14029 (2013).</li><li>Sharon, E., 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=Inferring+gene+regulatory+logic+from+high-throughput+measurements+of+thousands+of+systematically+designed+promoters.">Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters.</a> Nat Biotechnol. 30 (6), 521-530 (2012).</li></ol>

The authors have nothing to disclose.

O passo mais importante neste protocolo é design oligonucleotídeo adequada antes de pedir. Os oligonucleótidos devem ter as extremidades coesivas apropriadas para adicionado extremidades 5 &#39;da parte superior e inferior cadeias para assegurar que GGA incorporação é possível. Além disso, é importante para mudar a orientação da terminadores virada para a esquerda (terminadores que param de transcrição na cadeia inferior) ao do direito virado (termina a transcrição na parte superior fio) terminadores porq...

Large synthetic biology projects necessitate the use of highly effective transcription terminators to help regulate gene expression. Identification of novel terminators requires bioinformatic analysis of novel genomes. However, as increasing amounts of bioinformatic software are developed, each with a unique algorithm utilized for prediction, more discrepancy between putative results occurs. Because this process is somewhat subjective and is done in silico, these predictions need biological confirmation.1 Additionally, the volume of putative terminators identified through in-silico analysis requires the use of cloning strategies that can be completed in a relatively short time frame. 
The PGR-Blue plasmid is a modification of the PGR plasmid that has been redesigned to use Golden Gate Assembly (GGA) to simplify the cloning procedure by allowing for all reaction steps to be simultaneously performed in one micro-centrifuge tube.2,3 Color selection was incorporated into the plasmid to increase the ease of identifying positive colonies. A successful ligation should be white/yellow in visible light and fluoresce green under blue (450 nm) or ultraviolet (UV) light when grown on plates containing arabinose. Because uncut pGR-blue contains a blue chromo protein (amilCP), colonies containing an unmodified plasmid are blue under visible light. This simplification along with the streamlined protocol allows researchers to proceed from bioinformatic identification to biological confirmation in three to four days. The design nature of this system can be beneficial both in the research lab and in educational settings.
The pGR-Blue plasmid allows for quantification of terminator strength.4 A single arabinose inducible promoter is used to produce green fluorescent protein (GFP) and red fluorescent protein (RFP). The terminator is cloned into the plasmid after the GFP sequence but before the RFP sequence, thus stopping the transcription of the RFP protein. The strength of the terminator is determined by the ratio of GFP produced to RFP produced. 
The Vision and Change5 report suggested that Science, Technology, Engineering and Math (STEM) education incorporate research based experiences into the classroom.6 However, this requires the development of protocols that can be done by students with limited skill sets in a defined time frame. While the protocol can be accomplished in as little as three days, it was also designed so that each major step could be accomplished in a separate weekly (2-3 hr) lab period to create a Course Research Experience (CRE). When used in this manner, the procedure will take between three and six weeks and is appropriate for both introductory and advanced courses in Genetics, Cell Biology or Bioinformatics.

<table><tbody><tr><td>pGR-Blue Plasmid</td><td>Addgene</td><td>68374</td><td></td></tr><tr><td>pGR-Plasmid</td><td>Addgene</td><td>46002</td><td></td></tr><tr><td>AeraSeal-(Sterile Sheets)</td><td>Excel Scientific</td><td>BS-25</td><td>Sterile Sheets only</td></tr><tr><td>10x T4 DNA ligase Buffer</td><td>NEB</td><td></td><td></td></tr><tr><td>BsaI-HF</td><td>NEB</td><td>R3535S</td><td>The non-HF enzyme will work but is less heat stable.&amp;nbsp;</td></tr><tr><td>NEB Golden Gate Assembly Mix</td><td>NEB</td><td>E1600S</td><td>Commerial Master Mix refered to in the protocol.</td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>Round Microcentrifuge Floating Rack</td><td>Nova Tech International</td><td>F18875-6401</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma Aldrich</td><td>A9518</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-(+)-Arabinose</td><td>Sigma Aldrich</td><td>A-3256</td><td>D-Arabinose will not induce the pBAD promoter</td></tr><tr><td>Luria Base (LB) - Broth, Miller</td><td>Sigma Aldrich</td><td>L1900</td><td></td></tr><tr><td>Luria Base (LB) - Agar, Miller</td><td>Sigma Aldrich</td><td>L2025</td><td></td></tr><tr><td>Tecan-Infinite M200 Plate Reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Mix &amp;amp; Go Competent Cells - Strain JM109</td><td>Zymo Research</td><td>T3005</td><td>Use company recommended transformation protocol</td></tr><tr><td>ApE: A plasmid editor-software</td><td>http://biologylabs.utah.edu/jorgensen/wayned/ape/</td><td></td><td></td></tr><tr><td>Tris-HCl, Molecular Grade</td><td>Promega</td><td>H5121</td><td></td></tr><tr><td>Sodium Chloride (Crystalline/Biological, Certified)</td><td>Fisher Chemical</td><td>S671</td><td></td></tr><tr><td>Comercial Oligonucleotide synthesis</td><td>Integrated DNA Technologies (IDT)</td><td></td><td>http://www.idtdna.com/site</td></tr><tr><td>Microtest Tissue Culture Plates - 96 well (Sterile)</td><td>Falcon</td><td>35-3072</td><td></td></tr><tr><td>mycobacteriophage &quot;Bernal13&quot;</td><td>Genebank</td><td>KJ510413</td><td></td></tr><tr><td>Nuclease Free Water</td><td>Integrated DNA Technologies (IDT)</td><td>IDT004</td><td></td></tr><tr><td>Sterile, L-shaped Hockey-Stick Cell</td><td>Life Science Products</td><td>6444-S1</td><td></td></tr><tr><td>Nano-Drop 2000c UV-Vis Spectrometer</td><td>Thermo Scientific</td><td>2000c</td><td></td></tr><tr><td>ARNold: a web tool for the prediction of Rho-independent transcription terminators.</td><td>http://rna.igmors.u-psud.fr/</td><td></td><td></td></tr></tbody></table>

rapid verification of terminators using the pgr-blue plasmid and golden gate assembly

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.

Este protocolo irá produzir células que contêm PGR-azul com um terminador ligado entre GFP e RFP usando Golden Gate Assembly (Figura 2). As colónias positivas contendo inserção ligado pode ser seleccionado com base na cor. À luz visível colónias positivas será branco / amarelo e falsos positivos irá produzir colónias azuis após 18-20 horas de incubação a 37 ° C (Figura 3). Apó...

Watch this Scientific Journal Video about Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly at JoVE.com

Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

Rápida Verificação de Exterminadores Usando o PGR Azul plasmídeo e Montagem Golden Gate

This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed ...

rapid-verification-terminators-using-pgr-blue-plasmid-golden-gate

Research

JoVE Journal

Biology

7.6K visualizações.  Ouachita Baptist University. This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

Vídeo: Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

Conjugadas acasalamento Os ensaios para a análise específica da sequência de proteínas de transferência envolvidos na conjugação bacteriana

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Genética

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

Um protocolo Universal para grande escala gRNA biblioteca de produção a partir de qualquer fonte de DNA

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: Uma alta taxa de transferência baseada no sequenciamento técnica para identificar a tarde replicando regiões do genoma

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Uma tela de 2-híbrido de levedura em lote para comparar as interações da proteína

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Bioquímica

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, expressão de tipo específico de célula em Arabidopsis thaliana mediada através de LhGR Trans-ativação

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

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.

Montagem rápida de construções multigênicas usando clonagem modular de golden gate

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

Bioengenharia

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.

Circuitos Digitais Gênicos Baseados em Sistemas CRISPR-Cas e Proteínas Anti-CRISPR

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

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Rato Genoma Engenharia Usando Nucleases Designer

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Biologia

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.

Geração de plasmídeo vectores que expressam proteínas marcadas-bandeira nos termos do regulamento de Alongamento Human Fator-1α Promotor Usando Assembleia Gibson

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

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.

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.

Strand DNA Deslocamento Portas para redes de reação de Execução químicos derivados do plasmídeo

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 ...