We thank C.C.R. laboratory members for comments and S. Moskowitz for figure preparation. This work was supported by National Institutes of Health Grants F32GM101761 (A.J.H.) and GM54397 (C.C.R.).

注:按照有关放射性物质的安全使用和处置的所有机构的规定，包括但不限于个人防护装备的使用，如手套，防护眼镜，白大褂，和适当的丙烯酸盾牌。 注:标准缓冲液是由40毫米的HEPES-KOH，pH值7.5，50毫米钾谷氨酸，5毫摩尔DTT，0.1毫摩尔EDTA（以5倍浓度此缓冲区是预先定制的），每个的dNTP为0.3毫米的。 T7复制蛋白质如所述8，9，10纯化。含有单一的T7引发酶识别位点（5&#39;-GGGTC-3&#39;，5&#39;-GTGTC-3&#39;，5&#39;-TGGTC-3&#39;）的单链DNA可以从各种不同的DNA合成公司（长度可购买的单链DNA可取决于所选择的酶，T7引发酶，五聚体是最小的模板长度以合成全长的引物11，作为隐蔽C是必不可少的，但是，如果该引物是要通过聚合酶延伸，额外的核苷酸必须出现在模板的3&#39;末端。反应通过加入10mM的MgCl 2的最终浓度的启动，并在25℃温育。人工取样工作得很好，5秒以上的时间点。如果需要的时间点比短，建议使用一个快速骤冷流速仪以获得准确的数据。 1.多成交引物合成反应由手工取样<ol><li>在实验开始前，试样2甲酰胺染料缓冲液（93％（体积/体积）甲酰胺，50毫摩尔EDTA，0.01％二甲苯蓝和0.01％溴酚蓝）转换成8 1.5毫升聚丙烯管中。在一般情况下，管的数目等于所分析的时间点的数量。 </li><li>制备20微升混合物由1×缓冲液，0.1mM的ATP和CTP，0.25毫居里/毫升[α-32 P] CTP，0.1μMSSDNA模板和0.1μMGP4（表示为六聚体浓度 - 0.6μM单体）。 </li><li>在室温下孵育该混合物5分钟。 </li><li>除去使用移液器混合物2微升并在步骤1.1为无反应控制（时间零）制备的1.5毫升管2微升甲酰胺染料缓冲器混合。 </li><li>加入2微升的0.1M的MgCl 2反应混合物（以10mM的最终浓度）。 </li><li>在10秒的间隔手动收回使用移液器反应混合物2μL样品，并在步骤1.1编写的1.5 mL管predispensed甲酰胺染料混合。 </li><li>在95℃下热样品在热块5分钟。 </li><li>加载样品的等分试样到变性凝胶。由T7启动引发酶合成的小tetraribonucleotide底漆产品上的0.4毫米解析以及厚25％的丙烯酰胺:双丙烯酰胺（19:1），3M尿素，1×TBE（100毫摩尔Tris-硼酸盐，1mM EDTA）中。 </li><li>放在整个chromatogra凝胶PHY纸，用保鲜膜覆盖。干凝胶使用凝胶干燥。 </li><li>制备系列稀释的反应混合物的剩余部分，并当场加载的凝胶上色谱纸在相同体积， 即 ，发现稀释液4微升如果样品的4微升装载。 注意:这将作为标准曲线来确定在反应过程中形成的产物的浓度。例如，使用的TE缓冲液（10mM的Tris-HCl，pH值8.0，1mM EDTA）中很好地工作，跨越一系列五倍稀释1:25到1:15625（5 2 -5 6），但稀释应根据活动的水平进行调整。 </li><li>揭露使用磷光屏干胶和标准。量化在稀释系列中的放射性和描述8构建的标准曲线，12 注:T7 DNA聚合酶的夹杂物向反应混合物允许一个EXA矿井tetraribonucleotide引物，通过这种酶的使用量。最佳地，聚合酶浓度应饱和简化数据的解释。 0.4微米或更高的聚合酶浓度给出了良好的效果。此外，可以以这种方式被检验其他蛋白质，如单链DNA结合蛋白，在任引物合成或滞后链引发的效果。 </li></ol> 2.多成交引物合成反应中，用快速淬火仪<ol><li> 反应混合物的制备 <ol><li>制备溶液A的400微升，含有1x缓冲液，0.2μM的GP4六聚体，0.2微米的ssDNA模板，0.6毫的dNTPs，0.2mM的ATP和CTP（含0.5毫居里/毫升[α-32 P] CTP。 </li><li>制备400μL的溶液B，含有1x缓冲液和20mM的MgCl 2。 </li></ol></li><li> 快速淬火流量仪表的操作 <ol><li>开启说唱ID-淬火流量仪表;出现主菜单之后，该仪器的键盘上键入7到注射器驱动器移动到原位置。 </li><li>打开缓冲注射器位置"LOAD"。 </li></ol></li><li> 装载缓冲注射器 <ol><li>附上含有缓冲驱动注射器端口10毫升的注射器。更改注射器旋钮位置"LOAD"。 </li><li>使用10毫升注射器，填仪缓冲液储A和B用10mM的Tris，pH值7.5，1mM EDTA和填充骤冷溶液（250毫摩尔EDTA和0.2％SDS）中注射器℃。通过在进出推动柱塞除去气泡。 </li><li>更改旋钮的位置，以"火"。 2型调整注射器驱动器杆的位置。按"向下调整"按钮，直到缓冲区出来的退出循环，以降低注射器（连续7-下降; 8大降压; 9小步向下）。键入"ESC"键，返回到主菜单。 </li></ol></li><li> 清洗油管 <ol><li> ATTACH出口管线至真空。更改样本旋钮"刷新"。由旋钮设定到水平位置附近的缓冲注射器。打开真空和浸2冲洗循环的H 2 O代表10秒，然后在甲醇浸10秒。干循环通过吮吸〜15秒的空气，或直至干燥。 </li></ol></li><li> 装载样品 <ol><li>更改样本旋钮位置"LOAD"。将溶液A到1毫升注射器。注射器插入到样品负荷的港口之一。重复溶液B，并在相反的样品装载口放置。 </li></ol></li><li> 的时间点采集 <ol><li>在主菜单中，对于淬火流运行1型。输入反应时间（s），然后按"ENTER"。反应循环使用数量将被显示。切换到所需的反应循环。在步骤2.2.4重复清洗过程。 </li><li>打开样品旋钮"LOAD"。负载反应样品混合循环直到刚刚中央搅拌室外面。将所有旋钮"FIRE"，确认所有的旋钮都在正确的位置。 </li><li>将1.5毫升管到出口环结束。按下"GO"，以运行反应。在步骤2.2.4重复清洗过程。刷新缓冲区注射器A和B，直到他们击中了司机。 </li><li>重复步骤2.2.6.2-2.2.6.3为每个所需时间点。例外:请勿将包含启动试剂（ 如 ， 氯化镁在这种情况下）淬火零时间点控制时的解决方案。 </li><li>当收集完样本，输入"ESC"键返回主菜单。按"7"到缓冲针筒驱动器返回到其初始位置。 </li></ol></li><li> 仪器清理 <ol><li>除去反应混合物注射器。打开样品装载旋钮"LOAD"位置。在真空下，洗净用10ml 0.2N的氢氧化钠每个样品环，接着用10毫升0.3的NH 3 PO 4和10ml MeOH中。 </li><li>打开注射器旋钮"LOAD"位置。向下按所有3个SYR英格柱塞检索反应和淬火缓冲区。洗涤用5毫升水2 O注射器至少两次。 </li><li>用5毫升水2 O再次填充注射器和转旋钮"火"的位置。改变旋钮位置并打开真空，除去H 2 O.作为冲洗步骤2.2.4。关闭仪器。 按照步骤1.7-1.12描述分析产品:注意 。此外，T7 DNA聚合酶和其它复制蛋白可以在反应混合物中加入以分析其对引物合成或扩展的效果。 </li></ol></li></ol> 3.单成交引物合成反应 注:单成交引物合成/扩展试验也同样进行了多次失误的反应，有几个主要区别:这些试验遵循放射性标记的ATP到底漆或延伸产品的转化，基板但是浓度和蛋白质considera布莱高。另外，为了达到毫秒的分辨率，以使引物合成反应的分析，必须利用一个快速淬火流的机器。 <ol><li> 反应混合物的制备。 <ol><li>准备溶液A 400微升，含有1x缓冲，3μMGP4六聚体，6μM单链DNA模板，0.6毫米的dNTP，1毫米CTP和2纳米[γ-32 P] ATP。制备400μL的溶液B，含有1x缓冲液和20mM的MgCl 2。 </li></ol></li><li> 快速淬火操作流程 <ol><li>进行反应，如步骤2.2所述。如果需要的话，添加T7 DNA聚合酶或T7单链DNA结合蛋白在15μM和20μM的浓度，分别。在这种情况下，延伸产物积聚在允许用于反应的手动采样时间制度。 </li></ol></li></ol> 4.数据分析<ol><li>配合产品进度曲线非线性least-使用市售软件能够最小二乘的配合和/或数值积分平方模型。操作的细节将随所使用的软件变化，但以下是用于拟合数据的方程的广义形式。 </li><li>通过拟合进步曲线的线性方程确定的产物形成多个周转引物合成反应的速率。如果有显著曲率，通过将数据拟合到单指数函数使用初始速率方法13，14:Y = A（五- K t），其中y是产物浓度，A是反应振幅，k被所观察到的速率常数，t是时间，单位为秒。在时间= 0的切线的斜率是初始速率。 注:飞度前稳态引物合成反应的线性方程或预稳态突发方程Y = A（E - K BURST T）+率SS t，其中A是反应幅度，K 爆是预稳态突发观察到的速率常数，速率ss是稳态率SS = K 猫 [酶]），t为时间（秒）。适合单成交引物合成反应发展到一个单一的指数函数y = A曲线（E - K T）。在GP4的情况下，通过数值积分15拟合数据，以具有三个NTP缩合步骤，其中每个中间是在与酶平衡模型，使用市售的软件。 </li></ol>

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The authors have nothing to disclose.

在执行这些实验的最重要的因素是高活性的纯化的酶的可用性。在我们与GP4的工作，例如，我们发现，在缓冲液中纯化的酶的含50％甘油的存储（20mM磷酸钾，pH 7.4，0.1毫摩尔EDTA，1毫摩尔DTT）在-20℃导致减少制备过几个月的比活性。因此，我们现在在25mM的Tris-HCl，pH值7.5，50 mM氯化钠，0.1毫摩尔EDTA，1毫TCEP纯化GP4，和10％甘油的闪冻小等份用液氮并将其存储在-80℃。在使用快速淬火流仪，特别是采?...

DNA的复制是所有细胞生命的一个保守的特征。而复制组件的特性和数量有很大的不同，一般机制跨越进化1共享。这里，我们描述了基于凝胶的，不连续的动力学实验，使用的放射性底物，旨在通过T7启动复制体的组分理解体外滞后链DNA合成的动力学。 T7噬菌体的复制机制是很简单的，仅由四种蛋白质（DNA聚合酶，GP5，其持续合成因子， 大肠杆菌硫氧还蛋白，双官能引发酶-解旋GP4，和单链DNA结合蛋白，GP2的。 5）2。这一特性使得它来研究参与DNA复制保守的生化机制有吸引力的模型系统。 特别强调的是由T7 DNA形成的核糖核苷酸引物引发酶，一个critica升步骤中的DNA合成的起始。此外，还可以通过将其包括在反应混合物中检查这些引物由T7 DNA聚合酶或其他复制蛋白的用法。 T7启动引发酶 - 解旋酶，GP4，催化的引物DNA的合成在称为引发酶识别位点，或者PRS特定的DNA序列的形成，（5&#39;-GTC-3&#39;）。在PRS胞嘧啶是隐蔽，它是用于识别站点的必需的，但它不被复制到产品3。在引物合成的第一步由GP4涉及二核苷酸pppAC，然后扩展到三聚体，并最终官能四核苷酸引物pppACCC，pppACCA或pppACAC取决于序列在模板4的形成。这些引物然后可以通过T7 DNA聚合酶被用于启动DNA合成，这也是一个GP4辅助过程5,6。在这方面，引发酶域稳定极短的tetraribonucleotides与模板，防止其分解，以有利于保护底漆/模板到聚合酶活性位点7的方式从事DNA聚合酶。这些步骤（RNA引物的合成，引物越区切换到聚合酶和扩展）中重复多次循环复制后随链，必须与前导链的复制进行协调。 此处所描述的测定法是高度敏感的，并可以在一个温和的时间框架来执行。然而，它们是相对低的通量和必须十分小心以放射性物质的使用和处置行使。取决于在该反应进行时，人们可能会采用一种快速淬火仪在时间尺度适合用于在任一稳定或预稳态反应时间课程有意义的分析获得的样品的速度。最近，我们这里使用的描述测定提供我国证据的引物释放的从GP4的冈崎片段的开始时的引发酶域的重要性即此外，我们发现了为T7启动单链DNA结合蛋白，gp2.5调节作用的证据，在促进高效引物形成和利用8。

<table><tbody><tr><td>Tris pre-set crystals&lt;br/&gt; pH 7.5</td><td>SIGMA</td><td>T4128</td><td></td></tr><tr><td>Tris base</td><td>SIGMA</td><td>93362</td><td></td></tr><tr><td>L-Glutamic acid potassium salt monohydrate</td><td>SIGMA</td><td>G1501&amp;nbsp;</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Mallinckrodt Chemicals</td><td>5958-04</td><td></td></tr><tr><td>1,4-Dithiothreitol</td><td>J.T. Baker</td><td>F780-02</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>MP Biomedicals</td><td>811030</td><td></td></tr><tr><td>(Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate</td><td>Mallinckrodt Chemicals</td><td>4931-04</td><td></td></tr><tr><td>[&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P] CTP</td><td>PerkinElmer</td><td>BLU508H</td><td>radioactive, take protective measures</td></tr><tr><td>[&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P] ATP</td><td>PerkinElmer</td><td>BLU502A</td><td>radioactive, take protective measures</td></tr><tr><td>100 mM dATP, dCTP, dGTP, dTTP&amp;nbsp;</td><td>Affymetrix</td><td>77100</td><td>four dNTPs part of set</td></tr><tr><td>100 mM ATP and CTP</td><td>Epicentre</td><td>RN02825</td><td>part of NTP set</td></tr><tr><td>ssDNA constructs</td><td>Integrated DNA Technologies</td><td>N/A</td><td>custom sequences&amp;nbsp;</td></tr><tr><td>methanol&amp;nbsp;</td><td>Macron Fine Chemicals</td><td>3017-08</td><td></td></tr><tr><td>formamide</td><td>Thermo Scientific</td><td>17899</td><td></td></tr><tr><td>bromophenol blue</td><td>SIGMA</td><td>B8026&amp;nbsp;</td><td></td></tr><tr><td>cylene xyanol&amp;nbsp;</td><td>SIGMA</td><td>X4126&amp;nbsp;</td><td></td></tr><tr><td>acrylamide</td><td>SIGMA</td><td>A9099</td><td>Toxic</td></tr><tr><td>&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-Methylenebisacrylamide</td><td>SIGMA</td><td>M7279&amp;nbsp;</td><td>Toxic</td></tr><tr><td>Urea</td><td>Boston Bioproducts</td><td>P-940</td><td></td></tr><tr><td>Boric acid</td><td>Mallinckrodt Chemicals</td><td>2549</td><td></td></tr><tr><td>Rapid Quench-Flow Instrument&amp;nbsp;</td><td>Kintek Corp.</td><td>RQF-3&amp;nbsp;</td><td></td></tr><tr><td>Kintek Explorer</td><td>Kintek Corp.</td><td>N/A</td><td></td></tr><tr><td>Kaleidagraph&amp;nbsp;</td><td>Synergy Software</td><td>N/A</td><td></td></tr></tbody></table>

kinetics of lagging-strand dna synthesis in vitro by the bacteriophage t7 replication proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

在协议中的步骤1， 即所述，通过手动采样多个周转条件下，通过GP4催化的引物合成反应获得图1A所示的结果。这里，凝胶电泳后的范围内的产品可以通过使用在引物的合成反应（ 图1A）的α位与32 P标记的CTP进行观察。标记的前体，CTP，显示出最高的流动性和向凝胶的底部，接着通过GP4合成对应于二，三较慢迁移的物种?...

Watch this Scientific Journal Video about Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins at JoVE.com

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

滞后链DNA合成的动力学研究<em&gt;体外</em&gt;由T7噬菌体复制蛋白

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

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Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

DNA Replication

SCIENTISTS IN ACTION

7.3K 次观看.  Harvard Medical School. We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7. 

视频: Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

科研

生物化学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

生物学

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA polymerase (RNAP) following a head-on collision is unknown. Here, we find that the E. coli replisome stalls upon collision with a head-on transcription complex, but instead of collapsing, the replication fork remains highly stable and eventually resumes elongation after displacing the RNAP from DNA. We also find that the transcription-repair coupling factor, Mfd, promotes direct restart of the fork following the collision by facilitating displacement of the RNAP. These findings demonstrate the intrinsic stability of the replication apparatus and a novel role for the transcription-coupled repair pathway in promoting replication past a RNAP block.

The fate of the replisome following a collision with a head-on RNA polymerase (RNAP) is unknown. We find that the replisome stalls upon collision with a head-on RNAP, but resumes elongation after displacing the RNAP from DNA. Mfd promotes replication restart by facilitating displacement of the RNAP after the collision.

复制叉停滞了迎头RNA聚合酶直接重新启动

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

生物工程

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

以此 T7 噬菌体的定量 PCR 研究

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

免疫与感染

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

寡核苷酸的化学三磷酸化

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

使用 T4 噬菌体探索小鼠模型中的肠道噬菌体-细菌动力学

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host&#39;s health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli,&#160;in the context of the murine gastrointestinal tract. The methods described here outline&#160;how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels.&#160;This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

作者聚焦:研究噬菌体诱导的 Gnotobiotic 小鼠免疫反应

Bacteriophages (phages), viruses that infect bacteria, are an integral component of the gut microbiome. Though these symbiotic inhabitants drive bacterial fitness and population dynamics, little is understood about how they impact gut homeostasis and disease. This protocol studies isolated T4 phages within a mouse model, adaptable to other phage-bacterial pairs.

小鼠肠道中 T4 噬菌体和 大肠杆菌 相互作用:研究体内宿主-噬菌体动力学的原型模型

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across ...

滞后链DNA合成的动力学研究

摘要

探索更多视频

此视频中的章节

DNA序列识别利用高通量原酶分析的DNAPrimase

可视化的单分子荧光显微镜下的DNA复制

使用单分子DNA拉伸含量直接观察DNA复制的酶

复制叉停滞了迎头RNA聚合酶直接重新启动

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

以此 T7 噬菌体的定量 PCR 研究

寡核苷酸的化学三磷酸化

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型

滞后链DNA合成的动力学研究

摘要

探索更多视频

此视频中的章节

DNA序列识别利用高通量原酶分析的DNAPrimase

可视化的单分子荧光显微镜下的DNA复制

使用单分子DNA拉伸含量直接观察DNA复制的酶

复制叉停滞了迎头RNA聚合酶直接重新启动

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

以此 T7 噬菌体的定量 PCR 研究

寡核苷酸的化学三磷酸化

小鼠肠道中 T4 噬菌体和 大肠杆菌 相互作用:研究体内宿主-噬菌体动力学的原型模型

小鼠肠道中 T4 噬菌体和 大肠杆菌 相互作用:研究体内宿主-噬菌体动力学的原型模型

小鼠肠道中 T4 噬菌体和 大肠杆菌 相互作用:研究体内宿主-噬菌体动力学的原型模型

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型