这项研究是由来自加拿大的自然科学与工程理事会授予发现（NSERC）的支持。

<ol>
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作者宣称，他们没有竞争的经济利益。

接合方法提供通过该细菌可以传播基因在挑战性的环境，如抗生素抗性标记的蔓延生长提供进化优势的装置。因为许多接合型质粒的是如此之大，在通过靶基因的突变对接合型质粒本身涉及的传送装置的组件中的蛋白12的功能的研究是非常笨重。本文详述的协议提供了通过其中一个可以更容易地通过使用更小，更容易管理的表达质粒（ 图1）的评估的目的靶基因的方法。我们采用在F质粒衍生pOX38-TC（ 表1）研究˚F质粒介导的结合;其他接合质粒可以使用这里详细的协议和相应的衍生质粒进行研究。概述的交配实验已经改编自弗罗斯特和他的同事，42与一些modificati附件。在以前的研究中，8,33,42所述的pOX38锝Δgene::厘米构建体的创建是通过用合适的限制性酶切割的感兴趣的基因和插入扩增的CAT盒插入pOX38锝实现。 42在目前的方法，我们采用的重组工程大肠杆菌 DY330R 33,34同源重组敲除的靶基因，并与CAT磁带替换它。这具有允许所得DY330R应变窝藏pOX38锝Δgene::厘米构造充当用于特定基因敲除的控制的优点进行的F-T4SS介导的经由传输使用pK184基因恢复质粒回收接合转移。而有可能产生使用CRISPER-Cas9方法，43击倒我们没有在这个时候探讨这种可能性。 该过程的F衍生质粒pOX38锝基因敲除的产生开始（FIGURË1）。这是通过在DY330R细胞同源重组（ 表1）来实现，也可以使用具有类似的功能，如DY329，DY331和DY378其它菌株。引物最初设计PCR扩增从pBAD33质粒32对CAT盒和包含悬垂是特异于靶基因（ 表2）的碱;然后将PCR产物电穿孔到DY330R细胞携带pOX38锝。同源重组产生敲除质粒pOX38锝Δgene::其中CAT盒被插在帧中的靶基因内，有效地破坏T4SS组件和缀合，同时提供Cm抗性厘米。同时，靶基因的PCR从pOX38锝扩增并插入到小表达质粒;在这个协议中，我们使用pK184用于组成型表达，但如果需要，人们可以选择质粒与靶基因的诱导表达。然后pK184基因质粒转化入DY330R pOX38锝Δgene::厘米细胞，这些细胞随后被用作供体转移pOX38锝Δgene::厘米经由缀合构造成XK1200细胞交配测定。在交配试验中，供体和受体细胞是XK1200 pOX38锝Δgene::分别Cm和MC4100。在pK184基因恢复的质粒，以及靶基因（缺失或点突变）的系列的突变体，提供给供体细胞，以评估其恢复交配，并确定其效率的能力，用MC4100受体细胞（ 图2 ;表3）。 关键的步骤是利用适当的供体和受体菌株（ 表1），和所使用的引物的设计。对于设计各引物，有应该遵循的一般准则。而我们严格尝试用40-60％的GC含量遵守，这可能不总是可能的。在这样的情况下，它是实验者的判断来测试的引物瓦特第i GC含量稍低于或高于此范围。正向和反向引物的熔化温度（T M）必须是相似的，和退火温度（T A）的值应始终大于由2-5℃，用于PCR的Tm 值低。自由能可用于允许发夹开发应比T 一低得多，而均聚物和杂二聚化的Tm的必须非常低（低于-10千焦和30℃）。引物可以被设计为期望的探测缺失，插入或点突变。它当然是至关重要的是，所得的基因构建被放大并连接到在框架中，使得它被适当表达的恢复质粒。感受态细胞转化可通过电穿孔或热休克使用或电感受化学感受态细胞分别进行。我们发现，电是大型结构，如pOX38-TC和同源重组oligonucle更高效otide，而较小的表达质粒如pK184-TRAF可以容易地转化为利用热休克方法细胞。最后，要记住，会有在整个协议使用多种抗生素，因为这两个供体和受体菌株需要不同的电阻是从在接合和回收的质粒所用的那些不同，它是重要的。 除了此处所描述的配合检测技术，但是也有一些用于研究接合等方法，在他们的方法稍有不同。水平基因转移是一种工艺，其中的细菌的质粒转移到受体细胞，包括种间的收件人。 44达尔伯格和同事例如使用44接合确定物种间的基因水平转移的程度的研究。他们利用绿色荧光蛋白（GFP）的成克隆到KT2442细胞质粒掺入;染色体升在KT2442细胞交流I Q基因抑制GFP表达。当质粒携带绿色荧光蛋白被转移到一个物种不与lac I Q基因，荧光观察。 44尽管其限制，以提供在不同物种的蛋白质的特定功能时，种间偶联实验也可能会被加上这里提出以使对于不同物种之间的蛋白质-蛋白质相互作用的进化预测的协议。

基因和蛋白质的在微观有机体水平转移起着细菌生存和进化以及感染过程的核心作用。的DNA在细菌之间或细菌和小区之间交换可以通过转化，接合或载体转导来实现。 1,2-共轭是革兰氏阴性细菌如大肠杆菌之间缀合过程中的比较，以在转换和转导独特，DNA转移中的供体-控制的方式，由此复杂的高分子系统连接供体和受体细胞发生。偶联也是其中的细菌细胞与宿主细胞相互作用，以注入基因，蛋白质或化学品中的主机系统的最直接的方式。 3很多时候，这些药剂的转移在主机上拥有显着的效果，从发病机理和致癌主办进化和适应。它已经表明，共轭recombinatioN增加适应3倍的细菌具有高的突变率环境胁迫的条件下的速率。 4此外，缀合是迄今为止通过在细菌菌株的抗生素抗性基因被传播的最常见的途径。 5,6 微生物已经进化专门的分泌系统，以支持跨越细胞膜大分子的转移;目前有9种类型的革兰氏阴性细菌分泌系统（的TSS）的已经被描述:T1SS，T2SS，T3SS，T4SS，T5SS，T6SS，T7SS，以及上述Sec（分泌）和TAT（双精氨酸易位）通路。 7,8-每个型分泌系统的进一步分为不同的亚型，必然因蛋白质的多样性和所涉及的途径的独特性，在不同的细菌菌株。例如，在IV型分泌系统（T4SS），Ti和CAG系统便于执行传输而在F-质粒，R27一第二pKM101 T4SSs便于接合质粒转移。 7,9,10由生物体及其组成蛋白质组装各自的分泌系统和共享细胞内容与收件人或其周围环境的机制进行了详细的了解是有针对性的发展战略，以打击病原微生物的过程中的一个重要因素细胞感染。 以下在大肠杆菌中由莱德伯格＆塔特姆，11初始识别接合大量的移动和接合质粒已被鉴定和表征。 12这种移动质粒显示出相当大的范围是尺寸（从1至200千碱基（kb的）），但是所有移动质粒含有一个relaxase，其识别传送（ORIT）从而使质粒的传输的起源。接合质粒进一步编码基因的功能性T4SS的装配以及一型四耦合蛋白。 12例如大肠杆菌的100 kb的˚F质粒编码33.3 KB转移（TRA）区域内的所有的共轭基因。 13时接合质粒转移有利于形成菌毛所有的蛋白质，交配对形成（MPF），DNA转移和排除功能的F质粒编码的TRA区域的基因。 10,14,15知识的显著体可用于接合T4SSs，但详细介绍了共轭蛋白质和复合物的结构研究只是最近变得可用。 16 - 28 为了组装工艺接合的全面视图，需要突变接合转移蛋白分析详细的结构研究的耦合。这可以通过接合交配试验来实现。为F质粒的TRA区域内编码的每个蛋白质起着的F -介导的共作用njugation;因此，敲除/缺失转印基因将废除细胞（ 图1）的共轭的能力。而较小的移动质粒是更有利于标准的删除程序，对于较大接合质粒如F，基因敲除更容易通过同源重组，其中靶基因被替换为一个传送一个不同的抗生素抗性基因来实现的。在目前的协议中，我们采用同源重组，以取代在55 kb的质粒˚F衍生pOX38锝氯霉素乙酰转移酶（CAT）的兴趣转移基因; 29,30所得敲除质粒pOX38锝Δgene::厘米，有利于耐氯霉素中生长培养基的存在下（厘米）。供体细胞窝藏pOX38锝Δgene::厘米不能影响通过使用检测交配观察接合DNA转移/交配;一个pOX38锝Δgene::厘米施主小区的配合效率和正常RECIPient将减少，或者更多的时候，被废除。在pOX38锝Δgene::厘米质粒的接合转移可以通过一个小复苏质粒窝藏转移靶向基因来恢复。此恢复的质粒可以是一个能够提供组成型表达，如质粒pK184（pK184基因），31个或一个，只要该质粒正确靶向基因到细胞（细胞质或周质）内的正确位置提供了诱导型表达。因此，在这种新的供体之间的配合测定（窝藏pOX38锝Δgene::厘米+ pK184基因质粒） 和受体细胞，匹配效率有望恢复到接近一个正常的供受体匹配检测的。这个系统使得一个通过一系列pK184基因构建体（缺失或点突变）的产生来探测敲除基因的功能和测试每个构建体的还原pOX38锝Δgene::厘米窝藏的配合能力的能力供体细胞秒。

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">GeneJet Plasmid Mini-Prep Kit</td>
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			<td style="width:212px;">Cuvettes have a 1 mm gap.</td>
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			<td height="21" style="height:21px;width:255px;">AvaI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0152S</td>
			<td style="width:212px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EcoRI</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">HindIII</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0104L</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NdeI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0111S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phusion DNA Polymerase</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">M0530L</td>
			<td style="width:212px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">T4 DNA Ligase</td>
			<td style="width:165px;">New England Biolabs</td>
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			<td height="21" style="height:21px;width:255px;">Chloramphenicol (Cm)</td>
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			<td style="width:131px;">BP904-100</td>
			<td style="width:212px;">20 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Kanamycin (Km)</td>
			<td style="width:165px;">BioBasic Inc.</td>
			<td style="width:131px;">DB0286</td>
			<td style="width:212px;">50 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nalidixic acid (Nal)</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:131px;">N8878-25G</td>
			<td style="width:212px;">10 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Rifampicin (Rif)</td>
			<td style="width:165px;">Calbiochem</td>
			<td style="width:131px;">557303</td>
			<td style="width:212px;">20 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tetracycline (Tc)</td>
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			<td style="width:131px;">BP912-100</td>
			<td style="width:212px;">10 &#181;g/mL</td>
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			<td height="22" style="height:22px;width:255px;">Streptomycin (Sm)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP910-50</td>
			<td style="width:212px;">50 &#181;g/mL</td>
		</tr>
	</tbody>
</table>

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.

˚F质粒驱动接合的过程中是一个协调的过程，涉及到的F质粒即组装一T4SS便于菌毛的合成和接合DNA转移的TRA区域内转移蛋白。蛋白质TRAF（GenBank登录＃BAA97961;的UniProt编号P14497）所需的接合的F-菌毛形成。 10,14,35 - 37蛋白质包含一个C-末端硫氧还蛋白样结构域，但它不具有催化CXXC基序。 35,38虽然已预测到通过其N-末端结构域的TraH蛋白质相互作用，如图39的区域比其C-末端结构域更动态，37否则不被知道关于该蛋白质的结构特征。为了评估与结构研究的结合TRAF蛋白质的功能方面，我们首先通过同源击倒了TRAF基因上pOX38锝重组，产生在大肠杆菌 DY330R细胞pOX38锝ΔtraF::厘米质粒（ 表1）。 33,34还产生来自TRAF特异性引物（ 表2）的pK184-TRAF质粒以提供偶联的回收和使探测蛋白质的序列。所述pOX38锝ΔtraF::厘米质粒转移到XK1200单元40从DY330R细胞时pK184-TRAF 在反式提供了（转导10微克/毫升捺生长，10微克/毫升Tc和20微克/毫升厘米）表示是:（a）在pOx38锝ΔtraF::厘米TRAF淘汰赛提供了一种在框架的CAT盒，和（b），该pK184-TRAF可以恢复共轭的功能。 采用采用XK1200 pOX38锝ΔtraF::厘米+ pK184-TRAFΔX的共轭匹配法37分析产生一系列的TRAF突变细胞和细胞MC4100 41分别为供体和受体。一个代表性突变体是TRAF 55-247（ 表1）中，N末端缺失突变体，去除该蛋白质的区域的预测与TraH交互。当全长TRAF蛋白被提供给供体细胞，接合转移被恢复（ 图2），同时提供空质粒pK184不（ 见表3）。同样，当用质粒pK184-TRAF 55-247（ 表3）设置在XK1200供体细胞接合功能没有恢复。这表明，该蛋白质的截短区域是接合装置内TRAF的功能是重要的，可能是通过与TraH相互作用，并提供了该蛋白质的区域为目标进行进一步突变分析。 <img alt="图1" src="/files/ftp_upload/54854/54854fig1.jpg" /> <s仲&gt;图1:共轭交配试验的示意图。在步骤1中，目标基因通过同源重组在DY330R细胞，一个有效的重组菌株淘汰，用PCR产生与突出端同源的靶基因的CAT盒。所得DY330R克隆携有质粒pOX38锝Δgene::厘米，这是无法除非敲除基因在反通过回收质粒（pK184基因）提供，以促进结合。所得pOX38锝Δgene::厘米质粒转移至XK1200应变（步骤2），用于使用交配测定用MC4100收件人（步骤3）的基因的进一步评估。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54854/54854fig1large.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/54854/54854fig2.jpg" /> 图2: 垫ING检测，以评估结合靶基因的功能。供体和受体细胞大肠杆菌 XK1200 pOX38锝ΔtraF::分别厘米转化有pK184-TRAF和MC4100。由此产生的接合子（MC4100 pOX38锝ΔtraF::厘米）生长在含Sm和CM平板，说明接合功能成功恢复。实验一式两份在单一琼脂平板上，并连续稀释以计算恢复性基因的突变体（ 表2）的配合效率使用。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54854/54854fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 细菌菌株/质粒 </td><td> 相关特性 </td><td> 选择性标记（S）† </s仲&gt; </td><td> 参考 </td></tr><tr><td> 菌株 </td><td></td><td></td><td></td></tr><tr><td> DY330R </td><td> W3110ΔlacU169gal490λc1857Δ（CRO-的bioA）里夫- [R </td><td>里夫</td><td> 33,34 </td></tr><tr><td> XK1200 </td><td> 的 F - lacΔU124Δ（NADA aroG加仑attλ生物的gyrA） </td><td> NAL </td><td> 40 </td></tr><tr><td> MC4100 </td><td> araD139Δ（ARGF-LAC）U169 rpsL150 relA1 flbB3501 deoC1 ptsF25 rbsR </td><td>钐</td><td> 41 </td></tr><tr><td> 向量和结构 </td><td></td><td></td><td></td></tr><tr><td> pBAD33 </td><td>从对araBAD的下质粒表达</td><td>厘米</td><td> 32 </td></tr><tr><td> pK184 </td><td> 2.4 kb的克隆载体，P15A复制</td><td>千米</td><td> 31 </td></tr><tr><td> pK184-TRAF‡&lt;/ TD&gt; <td> ˚FTRAF从pOX38在pK184 </td><td>千米</td><td> 35 </td></tr><tr><td> pK184-TRAF 56-247 </td><td> ˚FTRAF AA 56-247从pK184-TRAF </td><td>千米</td><td></td></tr><tr><td> 接合质粒 </td><td></td><td></td><td></td></tr><tr><td> pOX38锝</td><td> IncFI，茶+，RepFIA +，女，迷你-TN的F1 HindIII片段</td><td>锝</td><td> 29,30 </td></tr><tr><td> pOX38锝ΔtraF::厘米</td><td> pOX38锝与CAT插入TRAF </td><td>锝厘米</td><td> 35 </td></tr><tr><td colspan="4"> †厘米，氯霉素;公里，卡那霉素;最终，萘啶酸;里夫，利福平; SM，链霉素; TC，四环素</td></tr><tr><td colspan="4"> ‡所有TRAF构建包含19个残基的前导序列，以确保本地化的周质空间。 </td></tr></tbody></table> 表格1: <st荣&gt;电子。大肠杆菌菌株和质粒 在这项研究中使用。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 构造 </td><td> 底漆* </td></tr><tr><td> TRAF-CM-对于</td><td> 5&#39;- GATCGAGGCTGGCAGTGGTATAACGAGAAAATAAATCCGAAGGA - CTGTGACG GAAGATCACTTC -3&#39; </td></tr><tr><td> TRAF-CM-启</td><td> 5&#39;- TCTTCAGAAACGTTCAGGAACTGTTTTGCCAGGTCGTCCT - CTTATTCAGGCGT AGCACCAG -3&#39; </td></tr><tr><td> pK184-TRAF-对于</td><td> 5&#39;- GAATTC TTTTTT -3 TATGAATAAAGCATTACTGCCAC" </td></tr><tr><td> pK184-TRAF-REV </td><td> 5&#39;- TTTTTT AAGCTT TAAAAATTGGGTTTAAAATCTTCAGAAA -3&#39; </td></tr><tr><td> pK184-TRAF 55-247 -对于</td><td> 5&#39;- TACG CATATG ATGGCCGCACTGCAGACGG -3&#39; </td></tr><tr><td> pK184-TRAF 55-247 </sub&gt; -REV </td><td> 5&#39;- TACG CATATG TCCTGACGCCGGAAAAATAAAGCAGCAGAGTAA -3&#39; </td></tr><tr><td colspan="2"> †表改编自伦托等。 2016年35许可</td></tr><tr><td colspan="2"> * TRAF悬垂区域斜体。限制酶位点加有下划线（HindⅢ位:AAGCTT，EcoRI位:GAATTC，NdeI位:CATATG） </td></tr></tbody></table> 表2:本研究中使用的引物列表。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 供体质† </td><td> 回收质粒 </td><td> 结合子‡§（细胞毫升-1） </td><td> 配合Efficiency§|| </td></tr><tr><td> pOX38锝ΔtraF::厘米</td><td>没有</td><td> 0 </td><td> 0 </td></tr><tr><td> pOX38-TçΔtraF::厘米</td><td> pK184 </td><td> 0 </td><td> 0 </td></tr><tr><td> pOX38锝ΔtraF::厘米</td><td> pK184-TRAF </td><td> 5×10 3 </td><td> 0.0167 </td></tr><tr><td> pOX38锝ΔtraF::厘米</td><td> pK184-TRAF 55-247 </td><td> 0 </td><td> 0 </td></tr><tr><td colspan="4"> *表改编自伦托等。 2016年35许可</td></tr><tr><td colspan="4"> †供体细胞是大肠杆菌 XK1200，以3×10 7细胞毫升的平均浓度-1 </td></tr><tr><td colspan="4"> ‡收件人细胞大肠杆菌 MC4100。转导结合为阳性对照的数量是从10-5稀释。 0表示没有从10-2稀释转结合。 </td></tr><tr><td colspan="4">对于每个构建体进行的两到四个交配实验§An平均。 </td></tr><tr><td colspan="4"> ||交配效率defin编按100供体细胞转导。 0交配效率表示没有从10-2稀释转导</td></tr></tbody></table> 表3: 由TRAF缺失构建废止交配效率。

Watch this Scientific Journal Video about Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation at JoVE.com

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

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 ...

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13.7K 次观看.  York University. 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. 

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

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

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The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

诱导体细胞行业分析（ISSA）的为研究木材形成和二次开发茎相关基因和推动者使用

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

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

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

细菌结合频率的高分辨率比较

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

基于交配的酵母超表达文库筛选

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

基于细胞的 sineup 非编码 rna 检测, 可专门增强 mrna 的翻译能力

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

使用乌斯蒂拉戈梅迪斯作为特洛伊木马在现场交付玉米蛋白

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.