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>

<ol>
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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. 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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-蓝测试终止子。标准的经济结构由物资采购在实验室8间，然后混合。第二个协议使用商用预制的GGA预混。虽然这两种工作良好;商业主结构是更多的时间效率，但相比标准混合相对昂贵。在一个教育环境中，可能更经济，使标准GGA混合提前，并冷冻供学生个人用等分试样。 作为改性PGR为GGA并在选色相反方向加入amilCP的结果，绿色荧光蛋白和表达RFP的未切割的PGR-蓝质粒被限制并确定相对终止子强度时不应该被用作参考对照。然而，所有的修改都在克隆过程中，允许相对于终止子强度的适当的量化除去。虽然最好是使用原始未修改PGR质粒类似的结果可以通过克隆已知不被终止子成PGR-蓝色的顺序来实现。两个控件显示GFP的相等的水平和RFP的表达（ 图5），更重要的是，也有类似的GFP / RFP比值。 对于基因表达的调节紧张时，PGR-蓝质粒组成性表达阿糖胞苷调节所述的pBAD（阿拉伯糖诱导型）启动子。10,11取决于细胞系中使用的高阿拉伯糖浓度可能需要的GFP和RFP的表达。使用在大肠杆菌的10mM阿拉伯糖的浓度了良好的结果取得大肠杆菌株JM109。转化细胞应该是白色/黄色或可见光下的蓝色。在UV光下或蓝色光（450纳米），是很常见的只看到绿色荧光但在正转化的细胞而不是红色。然而，使用读板器检测RFP荧光。 作为概念证明该协议的终止符在分枝杆菌Bernal13进行鉴定。然而，PGR-蓝只能在E.大肠杆菌和它是不是可以在其他物种的细菌中表达的穿梭载体。其结果是，我们不知道究竟是如何确定的终结将在其主机环境中发挥作用，是一个潜在的局限性。表达盒准ð与PGR-蓝是由传统的克隆位点侧接，并且可以移动到一个适当的穿梭载体。这将允许个人研究者PGR-蓝定制他们的特定细菌菌种来说，如果需要的话。 虽然有其他的高通量测序分析程序使用，使用最多的一个正向遗传学方法。这些方法使用位点定向诱变或寡聚合成以产生数百至数千潜在序列的组合。12,13一旦创建，每个序列修饰的效果然后编目。然而，PGR-蓝是专为一个更直接的反向遗传学应用。当分析在硅片的新型基因组中，这是常见的在给定的区域来观察多个潜在终止子。 PGR-蓝旨在帮助研究人员很快被允许他们体内试验的推定序列完善其在硅片预测。应当指出的PGR终止测试plasmi天得到了用于定量终结者力量582序列4，但是PGR采用了限制性酶组装标准（ 例如 ，基本生物砖组件（BBA）），一个比较费时克隆过程相比，GGA。7 PGR-蓝质粒被设计为功能类似于PGR，但使用更快速的克隆程序和菌落颜色选择。这些修改，我们简化协议一起，使PGR-蓝适应教育和研​​究型实验室。

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 border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">pGR-Blue Plasmid</td>
			<td style="width:243px;">Addgene</td>
			<td style="width:115px;">68374</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pGR-Plasmid</td>
			<td>Addgene</td>
			<td style="width:115px;">46002</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AeraSeal-(Sterile Sheets)</td>
			<td>Excel Scientific</td>
			<td>BS-25</td>
			<td style="width:235px;">Sterile Sheets only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X T4 DNA ligase Buffer</td>
			<td>NEB</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BsaI-HF</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td style="width:235px;">The non-HF enzyme will work but is less heat stable.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">NEB Golden Gate Assembly Mix</td>
			<td>NEB</td>
			<td>E1600S</td>
			<td style="width:235px;">Commerial Master Mix refered to in the protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Microcentrifuge Floating Rack</td>
			<td>Nova Tech International</td>
			<td>F18875-6401</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">L-(+)-Arabinose</td>
			<td>Sigma Aldrich</td>
			<td>A-3256</td>
			<td style="width:235px;">D-Arabinose will not induce the pBAD promoter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Broth, Miller</td>
			<td>Sigma Aldrich</td>
			<td>L1900</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Agar , Miller&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L2025</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tecan-Infinite M200 Plate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mix &#38; Go Competent Cells - Strain JM109</td>
			<td>Zymo Research</td>
			<td>T3005</td>
			<td style="width:235px;">Use company recommended transformation protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ApE: A plasmid editor-software</td>
			<td style="width:243px;">http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl, Molecular Grade</td>
			<td>Promega</td>
			<td>H5121</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Sodium Chloride (Crystalline/Biological, Certified)</td>
			<td>Fisher Chemical</td>
			<td>S671</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Comercial Oligonucleotide synthesis</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td style="width:235px;">http://www.idtdna.com/site</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Microtest Tissue Culture Plates- 96 well (Sterile)</td>
			<td>Falcon</td>
			<td>35-3072</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mycobacteriophage &#34;Bernal13&#34;</td>
			<td>Genebank</td>
			<td>KJ510413</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease Free Water</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>IDT004</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, L-shaped Hockey-Stick Cell</td>
			<td>Life Science Products</td>
			<td>6444-S1</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nano-Drop 2000c UV-Vis Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>2000c</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">ARNold: a web tool for the prediction of Rho-independent transcription terminators.</td>
			<td style="width:243px;">http://rna.igmors.u-psud.fr/</td>
			<td></td>
			<td style="width:235px;"></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）中的分枝杆菌Bernal13进行生物信息学鉴定。 图4中的结果表明，用表达未切割/未修饰的PGR作为对照的细胞的相对终止子强度。如果预测的终止子具有类似于PGR一个GFP / RFP比值（〜0.0）或低于0.1，则该序列被确定为不是一个终止（T5和T6）。然而，每个数字增加第十代表终结强度相对于控制倍。例如，T3有GFP表达相对于RFP的高比例，并作为与对照相比，被认为是一个强终止子（ 图4）显示出在终止子强度的5倍的增加（0.5上图）。数据被收集在一式三份，平均为视觉表示。 要确定定量终结者实力的最佳参考对照，我们比较使用不含终止和PGR-蓝已知不是一个终止序列克隆原来的PGR质粒的原始数据。两个控件显示GFP和RFP的表达（ 图5）和，相似水平更重要的是，产生了类似的GFP / RFP比值。 <img alt="图1" src="/files/ftp_upload/54064/54064fig1.jpg" /> Figure 1.小号电气原理详图平板阅读器布局，房间应包括在酶标仪为三个独立的控制（绿色）。良好（1）含有LB +安培/阿拉伯糖肉汤只和未转化的感受态细胞（空白）井（2）作为一个空白。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）。在该板（黄色）的剩余部分可以被用来测试推定的终止子。建议所有的细胞，一式三份进行培养，并分析前的平均值。 <a href="https://www.jove.com/files/ftp_upload/54064/54064fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54064/54064fig2.jpg" /> 图2:克隆网站的定位及表达PGR-蓝。AmilCP的组成型表达（蓝蛋白染色体）被GFP和RFP的相反（下链）的方向推动。当用BsaI，AmilCP和BsaI识别位点消化除去和要测试的终止子被永久地连接到质粒。顶链和与适当的粘性末端终止子的底链被示出。该阿糖胞苷调节器和氨苄青霉素抗性基因的质粒，但没有显示。 <a href="https://www.jove.com/files/ftp_upload/54064/54064fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54064/54064fig3.jpg" /> 图3.后菌落筛选克隆。（A）PGR-蓝/终止结扎（标准混合）和细胞转化后，代表板块。蓝色菌落代表不成功结扎和白/黄山坳onies（由箭头指示）是成功的结扎。 （B）在展示改造后的代表殖民地同板块的图像缩放。背景颜色被改变的数字进一步突出菌落颜色差异。转化细胞（ 大肠杆菌 -JM109）接种在含氨苄青霉素和阿拉伯糖LB琼脂制地图。 <a href="https://www.jove.com/files/ftp_upload/54064/54064fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54064/54064fig4.jpg" /> 图4.六个不同的终结者在定量PGR-蓝六公认的终结（T1-6）中的分枝杆菌Bernal13进行生物信息学鉴定。结果表明相对荧光。使用含未切割/未修改PGR作为控制单元的所有结果正常化。 T3被认为是一个强有力的终止符。所有的结果代表一式三份个人成长殖民地和平均的视觉表现。误差棒代表平均数据点之间的偏差。 <a href="https://www.jove.com/files/ftp_upload/54064/54064fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54064/54064fig5.jpg" /> 图5. GFP和RFP表达的控制。GFP和RFP的未切割/未修改的植物生长调节剂和两种不同的非终止序列插入PGR-蓝表达。所有的结果代表一式三份个人成长殖民地和平均的视觉表现。误差棒代表平均数据点之间的偏差。 <a href="https://www.jove.com/files/ftp_upload/54064/54064fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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 San Sebastian

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

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

科研

生物学

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

快速PCR热循环利用微型热对流

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

快速合成及其与绿色荧光蛋白为基础的记者化学激活的转录因子筛选

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate 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.

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 ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...