作者要感谢马尔科姆·坎贝尔和托德Eckdahl与基因组协会的Active教学（GCAT）以及霍华德休斯医学研究所，科学教育联盟 - 噬菌体猎人推进基因组学和进化科学（SEA-噬菌体）计划。 这个项目是由国家研究资源中心（P20RR016460）和通用医学科学院卫生全国学院研究所（P20GM103429）资助。这项研究部分由美国国家科学基金会的资助下＃IIA-1457888的支持。此外机构（沃希托浸会大学）资金通过JD帕特森暑期研究奖学金提供。

1.设计和使用适当的粘性末端的寡核苷酸排序<ol><li>确定通过基因组分析使用的是免费提供的在线课程潜在的Rho-独立的终结。7 </li><li>当用双链DNA的工作，决定终止的取向来进行测试。7 PGR-蓝质粒只验证在5结扎&#39;到3&#39;的顶部（正向链）的方向终止子。 <ol><li>转换底部（反向）链终止其反向补充，重新调整顺序使用免费在线软件PGR-蓝测试， 如猿。 </li></ol></li><li>方向被确定为正确后，添加粘底"5&#39;-CGAC-3"&#39;5&#39;顶链的末端。的粘性末端"5&#39;-CCGC-3&#39;"添加到5&#39;和底链的末端。8这将确保连接的插入件将在适当的插入定向使用GGA。 </li><li>一旦序列测定，顺序单链寡核苷酸市场。 </li></ol> 2.退火寡核苷酸（仅适用于冻干DN​​A） <ol><li>重悬在无核酸酶的水个体寡核苷酸的100μM的浓度。 </li><li>使10×退火缓冲液:1M NaCl的和100mM的Tris-HCl pH 7.4的。退火缓冲液可以储存在4℃几周。一旦退火缓冲液是由和寡核苷酸重新悬浮，开始退火8。 </li><li>对于每个要测试的终止子，添加16微升的超纯水至1.5ml微量离心管中。 </li><li>加入2微升10X退火缓冲液中向每个离心管中。 </li><li>从期望终止子加入1μl顶链寡核苷酸（100μM）。 </li><li>从期望终止子加入1μl底链寡核苷酸（100μM）。 </li><li>创建沸水巴次使用含有在热板上大约400毫升自来水和地点的一个1000毫升烧杯中。 </li><li>将步骤2.6在浮动的离心管，煮4分钟。 4分钟后，关火板，但留下的管漂浮在水浴中缓慢冷却O / N（18小时），8。存放于-20℃退火终结。 </li></ol> 3.金门大会（标准混合 - 成本效率） <ol><li>使退火终结的40纳米的稀释。加入124μL核酸自由水至1微升退火终结混合（100微米）。 <ol><li>离心样品以10,000 xg离心30秒并储存在冰上。 </li></ol></li><li>为配合新的管适用于热循环，增加6μL核酸自由水。 </li><li>加入1μl商业DNA连接酶缓冲液[300mM的Tris-盐酸（pH值7.7-7.8），100mM的MgCl 2的，100毫摩尔DTT，和10mM ATP]。执行冰的所有后续步骤。 </li><li>加1微升PGR-蓝（35-50纳克/微升）的目标质粒的离心管。 </li><li>加入1μl的40纳米退火终结到离心管中。 </li><li>加入0.5微升商业高保真BsaI限制性内切酶的离心管中。 </li><li>加入0.5微升的商业DNA连接酶的离心管中。 </li><li>离心在10,000rpm xg离心120秒的反应混合物。然后直接放在管进入热循环。 </li><li>运行热循环程序:在37℃下20个循环1分钟，然后以1分钟，在16℃下，随后1个循环的15分钟，在37℃。使用的样品，立即冻结或并储存在-20°C。8 </li></ol> 4.金门大会（商业预混 - 时间效率） <ol><li>加入124μL核酸自由水的1微升100μM退火步骤2中创建这削弱了终止DNA浓度至40nm终结的。在执行新的这一步，标示1.5 ml离心管。 </li><li>进入一个新的适合于热循环管中，加入14微升核酸自由水。确保新管相应标记。 </li><li>加入2微升商业预混缓冲液的离心管中。 </li><li>添加PGR-蓝质粒1μL（35-50纳克/微升）的离心管中。 </li><li>加入2微升的40纳米退火终结到离心管中。 </li><li>加入1μl商业预混到离心管中。 </li><li>离心40纳米管在10000 XG 120秒。然后直接放在管进入热循环。 </li><li>运行热循环程序:在37℃1个循环的60分钟，随后1个循环的15分钟在55℃。使用的样品，立即冻结或并储存在-20°C。 </li></ol> 5.转型与殖民地的选择<ol><li>准备卢里亚基地（LB）琼脂含1毫米concentrat培养皿氨苄西林离子（AMP）和阿拉伯糖（ARB）的10毫米的浓度。制备至少10毫升1 M L -arabinose原液因为此储备溶液会在后面的步骤中使用。 </li><li>使用任何高效率（10月8日至十月九日转化子/微克）化学感受态大肠杆菌 （JM109）细胞转化。9为了达到最佳效果，加3-5微升连接反应至50μl感受态细胞。当使用化学感受态细胞，遵循针对正在使用的细胞转化协议。 </li><li>传播一半（25微升）的转化的细胞到预热的LB（安培/ ARB）琼脂使用无菌L形"曲棍球"平板上并在37℃孵育O / N。由于氨苄青霉素的分解的速度迅速，不于37℃经24小时孵育。 </li><li>根据通过目测颜色进行菌落筛选。一个成功的结扎应该是白色/可见光黄色和绿色荧光理解过程- [R蓝（450纳米）或紫外光。一个不成功的连接会产生一个殖民地是在18后在可见光下颜色为蓝色 - 20小时。 </li></ol> 6.核查和终结者的量化<ol><li>制备5-毫升无菌LB-肉汤管用1mM氨苄青霉素和10mM阿拉伯糖。需要管的数目是依赖于被测试的终止子加对照（细胞以氨苄青霉素抗性不发荧光和含有未切割PGR-蓝质粒的细胞）的数目。 </li><li>在可见光下使用挑无菌环2-3白色/黄色菌落（步骤5.4）。允许样品在肉汤，在37℃和160转16-18小时孵育振荡器上。不孵育这些样品在超过24小时。 <ol><li>任选地，使用分光光度计，以确定在600nm（OD 600）的光的细胞密度，以确保有足够的细胞生长。一般来说，〜0.8-1.0的OD 600后增长16-18小时的观察。 </li></ol></li><li>使用无菌96孔板的其余步骤。进行在无菌环境中，并以一式三份这些步骤。加入199微升无菌LB +安培/阿拉伯糖肉汤（1毫米氨苄西林/ 10毫米阿拉伯糖），每个井。包括在平板上三个独立控制的房间。 <ol><li>使用以下三个控件:只有一口井（1）含有LB +安培/阿拉伯糖肉汤和好（2）未转化的感受态细胞（空白）作为空白。用于定量（3）的终止子强度，含有未切割的原始PGR或含PGR-蓝与非终止序列连接单元应包括（为量化参考控制）。参见图1酶标仪布局。 </li></ol></li><li>吸管1μl的来自澳细胞溶液/ N肉汤到96孔板的各个孔中。 </li><li>密封板具有透气盖，摇晃在37℃18〜20小时，160转。 </li><li>使用读板器，通过读确定OD 600吸光度在600nm处。测量GFP荧光如下:激发395，发射509测量的RFP荧光如下:激发575，发射605 18-20小时后，将OD 600应为大约0.5。 </li><li>为了归在生长变化，使用的公式: <img alt="式（1）" src="/files/ftp_upload/54064/54064eq1.jpg" />对于GFP和RFP。 </li><li>标准化后，确定相对实力终止使用公式<img alt="公式（2）" src="/files/ftp_upload/54064/54064eq2.jpg" /></li></ol>

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	<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. 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The authors have nothing to disclose.

在这个协议中最重要的步骤是正确的寡核苷酸设计之前订购。寡核苷酸必须具有适当的粘性末端加入到顶部和底部链二者的5&#39;端，以确保GGA掺入是可能的。此外，为左朝向的终止子（终止子即停止对底链转录）的方向切换到右侧面对它是重要的（终止于顶链转录）终止子，因为GFP和RFP的表达是在正确的朝向（顶链方向）。 两个单独GGA协议和缓冲混合物可以用来在PGR-蓝测试?...

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.

该协议将产生含PGR-蓝用金门组装（ 图2）的GFP和RFP之间连接一个终止子的细胞。可以根据颜色来选择含有连接插入阳性菌落。在可见光阳性菌落将白/黄和假阳性将在37℃（ 图3）后培养18-20小时产生蓝色菌落。 菌落筛选后，读板器可用于确定终止子的强度。由于概念证明，六公认的终结（T1-6）中的分?...

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

终结者使用PGR-蓝质粒和金门议会的快速验证

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

视频: 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.

接合交配测定在接合介入转移蛋白的序列特异性分析

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

科研

遗传学

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.

一种从任意 DNA 源进行大规模 gRNA 库生产的通用协议

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: 一种基于高通量测序技术的基因组晚期复制区域识别方法

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.

酵母 2-混合筛在批次中比较蛋白质相互作用

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 ...

生物化学

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.

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

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.

使用模块化金门克隆的多基因结构快速组装

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

生物工程

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.

基于CRISPR-Cas系统和抗CRISPR蛋白的基因数字电路

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.

小鼠基因组工程使用Designer核酸酶

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

生物学

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.

质粒载体产生表达FLAG标记的蛋白质在人延伸因子1α启动子的使用吉布森大会规则

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.

质粒来源的DNA链置换盖茨实施化学反应网络

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 ...