这种材料基于工作支持的海军研究办公室奖号码 N00014-13-1-0074 （马路），人类前沿科学计划授予数量 RGP0037/2015年 （马路），和两国科学基金会授予 2014400 （马路）。

注意: 以下扩增步骤和提取方法在很大程度上可归纳为许多双链 DNA 噬菌体，例如， 噬菌体 T7、 肠杆菌噬菌体 T4 或肠杆菌噬菌体 λ (L)。它们主要用于噬菌体的基因组不是现成的商业来源购买。
 1.噬菌体扩增 注: 单斑块、 多周期 （次贷危机） 噬菌体生产技术是很好的描述为 T4 噬菌体在陈 等人 14 下面的噬菌体扩增和 DNA 提取方法是用 大肠杆菌 双链 DNA 噬菌体，例如，T7，T4，或者 L.推广议定书 》 的最终成功很大程度取决于所选的宿主细胞株 &#39; s 能够承受二重感染的条件。如果宿主细胞是不稳定的下二重感染，或从未到达二重感染，并裂解永远不会达到这一关键阶段，它是最佳着手汇合裂解协议。这包括分离通过高速离心和噬菌体的速度超速离心造粒的细胞碎片。所有以下条件和参数被作为一般的起始点。本地主机细胞系的最佳条件可能不同;确定和坚持适当的条件。
<ol><li>准备接种细胞 <ol><li>接种 10 毫升 Luria 贝塔尼 (LB) 媒体 15 毫升文化管与 大肠杆菌 宿主细胞，例如，B 或 K12。</li>
		<li>在激振器设置为 250 rpm 和 37 ° C.假一夜之间将增长至饱和。</li>
	</ol></li> <li>单斑块、 多周期噬菌体生长和浓度 <ol><li>准备一盘主管噬菌体斑块，温暖在 1 h，37 ° C 的几个 LB 琼脂平板或隔夜。</li>
		<li>准备几个稀释度的噬菌体股票 （例如， T7，T4 或 L） 的已知浓度在 LB 媒体，针对 ~ 10 2-10 3 噬菌体/mL。</li>
		<li>的隔夜细胞生长的集中的主机到每个板添加 100 微升。</li>
		<li>选择卷的准备的噬菌体稀释对应于范围从 10-100 噬菌体/板。将所需的卷的噬菌体稀释液添加到每个板 (例如，100 μ L 的 10 3 噬菌体/毫升; 这应该会产生 ~ 100 斑块)。孕育在 37 ° C 的板和开始向上计数定时器 （+ 0 h）。孵育 4 5 h.</li>
		<li>准备 49 mL LB 媒体在 250 毫升瓶和地方在旋振筛设置为 250 rpm 和 37 ° C.</li>
		<li>在 + 2.5 h，稀释细胞 50 x 从隔夜增长 49 mL LB 培养基预热瓶加 1 毫升的饱和的细胞培养。一旦细胞达到日志增长 （+ 4 ~ 5 个小时），测量吸光度在 600 毫微米 (OD 600)。确定细胞浓度的使用转换 OD 600 1.0 = 8 x 10 8 细胞/mL。</li>
		<li>摊薄到 2 x 10 7 细胞/毫升使用额外 LB 培养基。删除包含从孵化器的噬菌体斑块琼脂平板。立即使用无菌巴斯德吸管年底核心和删除单个的斑块。吹进稀释细胞的斑块和额外的 2 h （+ 6 7 h），37 ℃ 培养。</li>
		<li>充分考虑 500 µ L 样品到 1.5 毫升管、 添加 10 µ L 三氯甲烷 (氯仿 3)，并立即高速涡流感染试验。观察是否解决方案已经澄清。一旦完全感染细胞，孵育另一个 2 h （+ 8 9 h）。 
		注意: 如果单元格完全被感染了，他们迅速将溶解 (&#60; 2 分钟) 和澄清的溶液。其他成果被认为在 讨论 下面的 节 中。单元格将 superinfected，但不是会溶解到这一点。如果开始溶解，另外的头和收获用离心法必须立即开始以防止噬菌体退化。</li>
		<li>添加头 5 微克/毫升和离心机在 8,000 x g，4 ℃ 条件下，颗粒细胞。弃上清。重悬在 10 毫升的 1 x TRIS 氯化镁 (TM) （50 毫米为三，ph 值为 7.8，10 毫米氯化镁 2） 颗粒缓冲与 5 微克/毫升头。高的速度，以将细胞溶解添加 500 µ L 氯仿 3 和涡流。澄清用离心法在 10 分钟的 12,000 x g 4 ° C.既体面上清液进入 15 毫升锥形管和储存在 4 ° C.</li>
	</ol></li></ol> 2。纯化噬菌体 注: 蔗糖净化是很大程度上依赖于噬菌体的大小。噬菌体被孤立的大量的注意事项将必须作出进行梯度分离条件的调整。最后病毒滴度测定的超过 10 13 噬菌体/mL 是用这种方法很容易实现。
<ol><li>准备 12 毫升的 5%和 45 %w / v 蔗糖在 x TM 缓冲区 1。</li>
	<li>准备 4 x 5-45%蔗糖梯度由 45%蔗糖转化 4 离心管移液 2.5 毫升。与顶尖 ~ 2.6 毫升的 5%的蔗糖，停止当液体达到 2 毫米从管的 rim。 
	注意: 放置与蔗糖溶液表面接触针提示和非常慢慢地免除液体。较重，轻的蔗糖溶液会浮在上面的这将形成一个分明的界限。正确分层的解决方案将有 ~ 1 毫米接口，如果对背景光举行。</li>
	<li>混合梯度倾斜管轮流使用渐变成形仪 43 23 rpm 86 ° s。如果不立即需要用石蜡膜覆盖并存储在 4 ° C.</li>
	<li>从顶部的每个删除 500 微升准备 1 毫升滴与平衡向内 ± 0.002 g.蔗糖梯度</li>
	<li>从所有管 （步骤 1.2.9） 池噬菌体悬浮液。使用 1ml 移液器，引进来与液体表面接触提示和非常缓慢配药卷到蔗糖梯度，小心翼翼地不混合通过快速用移液器的顶部添加 500 µ L 的暂停。离心机在 70,000 x g 20 分钟在 4 ° C. 
	注意: 平衡准备的渐变到内 ± 0.002 g.小噬菌体可能需要高达 1 h.</li>
	<li>删除使用无菌注射器和钝器套管的噬菌体乐队。 
	注: 当从侧面观察，噬菌体乐队将厚和乳白色相比，周围的环境，在高度，大约 5 毫米，位于离心管的半路上。
	<ol><li>淹没钝插管到直到提示解决方案集中在噬菌体乐队非常上部边缘。通过在注射器柱塞上画画，噬菌体带绝大多数直至删除乐队。</li>
	</ol></li> <li>向 3 4 离心管中添加蔗糖卷与悬浮的噬菌体。添加不超过 1.5 毫升/管。~ 3 4 毫米自上而下与冷 1 管填充 x TM。用石蜡膜覆盖和反相混合。回到在离心和自旋为 1 h 在 4 ° C 145,000 x g以颗粒。较小的噬菌体可能采取达 2 h.</li>
	<li>快速倒出上清液和排泄倒在一次性湿巾。擦干用无菌棉签来删除多余的上清液管内部。</li>
	<li>分 200-400 µ L 冷 1 x TM 跨所有微丸和悬一夜之间在 4 ° C; 无抖动是必需。 
	注意: 如果小球是不干净的 例如， 粘稠位的膜，DNA、 等，游泳池和执行额外的离心离心 17,000 x g.</li>
	<li>使效价前, 一天准备 大肠杆菌 宿主细胞在 10 毫升 LB 培养基文化并留在一个颤抖的孵化器，一夜之间设置为 250 rpm 和 37 ° C。适量的至少 1 小时，37 ℃ 培养板孵育效价的噬菌体股票之前。 
	注意: 板孵化数取决于稀释度的噬菌体股票待镀数目: 噬菌体股票每个独特稀释需要两大文化板块 （例如，四个不同稀释度的噬菌体股票需要八个培养皿）。</li>
	<li>备 2.5 g 磅和 0.6 g 细菌琼脂中加入一瓶 100 毫升 100 毫升的顶级琼脂。溶解固体的去离子水 100 毫升和高压釜的卷中。在高压釜之后, 慢慢反转瓶 6-8 倍同质化琼脂整个解决方案。将瓶子放在 45 ° C 水浴为 15-20 分钟来平衡温度 .</li>
	<li>编写的 LB 解决方案的噬菌体股票连续稀释。
	<ol><li>添加 990 微升磅到 10 µ L 噬菌体为 100 稀释的的股票。同质化的旋涡。10 稀释因子，添加 100 μ L 的噬菌体股票到 900 µ L 磅涡同质化。</li>
		<li>重复获得数个斑块 （10-100 斑块） 需要多少倍以上稀释系列。 
		注意: 要实现这一目标，稀释噬菌体股票 2-3 个数量级小于预期噬菌体产量的噬菌体/mL 反应。例如，如果特定的噬菌体股票包含 10 11 传染性噬菌体/mL，印版噬菌体股票在 10 8-折或 10 9-折叠稀释。</li>
	</ol></li> <li>地区准备噬菌体效价的装配 14 毫升试管 （文化管数目等于噬菌体股票稀释待镀数目）。从步 2.12 的股票样品稀释噬菌体和宿主细菌细胞从步 2.10 置于冰</li>
	<li>准备主混合吹打 5.25 毫升顶级琼脂 （步骤 2.11），220 微升稀释噬菌体样品 （第 2.12 步），和 50 µ L 宿主细菌细胞 （步骤 2.10） 到 14 毫升文化管。帽的文化管和高速度的涡。
	<ol><li>将剩余的噬菌体解决方案存储在 4 ° C.</li>
	</ol></li> <li>检索两大文化板块 （步骤 2.10） 从 37 ° C 的孵化器。慢慢添加 2.5 毫升的主组合的第一板 （无气泡） 中心。轻轻旋转板用手均匀分布主混合，以使它跨越整个文化板块。重复第二板。等待 20 分钟，以确保最高琼脂凝固。孵育板在 37 ° C 的 4-7 h.</li>
	<li>计算斑块，并确定每个反应样品的噬菌体浓度. 
	注: 斑块会显示为 1-2 毫米明确圆圈在宿主细胞的不透明的地毯。代表的结果，请参阅 图 1。成功的噬菌体生产将导致最终浓度为 10 12-10 13 噬菌体/mL。</li>
</ol> 3。双链基因组 DNA 提取 注: 稀释，颤抖着，和离心步骤必须优化发展之间苯酚和水相中的又厚又结实的蛋白质边界层。这将生成最高纯度基因组来说，都是免费的蛋白质污染物。初始稀释是依赖最后噬菌体效价。极高滴度的噬菌体股票 (&#8805; 10 13 噬菌体/mL) 可能需要 10 ~ 20 倍稀释前提取，而低滴度股票 (~ 10 10-10 11 噬菌体/mL) 可能只需要 2 倍或根本没有。如果它很难在随后的步骤中形成固体蛋白质层或悬浮液是太黏要可行由于 DNA 浓度较高，可以考虑噬菌体股票继续提取之前高稀释。在任何基因组处理步骤中，它是重要时要使用大口径吸液管吹打任何水相中。多个噬菌体基因组都相当大，很容易分散通过吸管剪。此外，任何涡流应当明确地避免，因为这将严重剪切基因组。
<ol><li>稀释噬菌体股票从步 2.9 至 400 微升 1 x 中新鲜 1.5 毫升管 TM 缓冲区的一部分。</li>
	<li>Tris:Phenol:Chloroform 等体积加入稀释。震，混合物轻轻地对实验室的摇杆，或用手，5 分钟离心机在 5-10 分钟，台式离心机 17,000 x g 的合成乳胶 
	注: 底层的苯酚相表面不同，白蛋白层是本。</li>
	<li>删除到照顾不来打扰相界面的新管上部的水相。</li>
	<li>重复步骤 3.2-3.3 额外 2 次，共 3 酚提取。将水相转移到新的离心管和加同等体积的港务局 3。摇和离心机 （步骤 3.2）。转移到清洁管中的水的 DNA 样本。</li>
	<li>估计纯化 DNA 的量。添加 0.4 卷 3 M 醋酸钠 (CH 3 COONa) 和 95%乙醇 （乙醇） 的 3 卷。放置在-20 ° C 冰箱隔夜沉淀。离心机在 10-15 分钟的台式离心机 17,000 x g 
	注: DNA 立即脱水，并使其作为大众在管中的悬浮液中可见。适当的生成颗粒是白色和反对试管底部包装得很好.</li>
	<li>删除和弃上的清调迁或用移液器。添加 500 µ L 70%乙醇并轻轻摇动管，直到颗粒漂浮免费从管底。离心机在 17,000 x g 5 分钟删除并丢弃乙醇，照顾不打扰颗粒。</li>
	<li>重复步骤 3.6。空气干燥颗粒剂对台式为 30-60 分钟添加 50 µ L ddH 2 O 到小球和孵育 1 h 在室温下或在 4 ° C，重悬在一夜之间。确定 DNA 浓度使用吸收测量在 280 nm。 
	注: 预期的浓度为 0.5-5 微克/微升使用这种技术。除以总基因组分子重量来确定最终的基因组浓度。</li>
</ol> 4。无细胞噬菌体反应和噬菌体效价实验 <ol><li>准备 25 g LB 培养基和 15 g 细菌琼脂固体、 倒入 1 升瓶，和去离子的水添加 1 L.釜。</li>
	<li>后灭菌，反转瓶慢慢地 6-8 倍的照顾，避免泡沫的形成。瓶反演将同质化琼脂固体整个 1 L 溶液。文化盘子配药前 20 分钟 58 ° C 水浴中放置瓶子。</li>
	<li>一旦 LB 琼脂溶液的温度达到平衡，准备一束火焰旁边的培养板来消毒的开放培养板附近的环境。在水浴以免凝固的琼脂同时整除数保持 1 升瓶。100 毫米 x 15 毫米培养板中加入 25 毫升。1 L 将产生 40 培养板。</li>
	<li>拨出一个文化板块，让至少 1 小时，在室温下固化; 此板将用于电镀宿主细胞。让其他 39 文化板固化 2 天在室温存储在 4 ° C 或立即用于制作效价。</li>
	<li>由裸奔适当的细菌菌株 （如 主机板宿主细胞B 为 T7)，被储存在-80 ° C，到 LB 琼脂培养板上。条纹在明火，避免环境污染。培养过夜在 37 ° c。宿主细胞有没有抗生素的耐药性，因此在无菌的环境中工作至关重要。</li>
	<li>无细胞反应 
	注: 无细胞 TXTL 反应分为粗 大肠杆菌 提取 （8.9 9.9 毫克/毫升蛋白） 组成的反应缓冲区和噬菌体基因组的其他 67%33%。粗提物被准备像以前那样描述的 15 ， 16。最后反应条件是: 8.9 9.9 毫克/毫升蛋白 （从原油中提取），3-6 毫米毫克-谷氨酸，40-100 毫米 K-谷氨酸，2-4%PEG 8000，3-4 毫米的每个氨基酸，准备作为先前描述的 17 和能量的混合溶液，描述 15，以前组成 0.33 3.33 毫米 DTT，50 毫米复，1.5 毫米 ATP 和 GTP，0.9 毫米 CTP 和 UTP，0.2 毫克/毫升 tRNA、 0.26 毫米 CoA、 0.33 毫米 NAD、 0.75 毫米营、 0.068 毫米亚叶酸、 亚精胺 1 毫米和 30 毫米 3-PGA。噬菌体 DNA 类型需要 0.5-10 nM 基因组浓度和 RNA 型噬菌体需要 50-150 毫微米范围。最后反应浓度为合成特定的噬菌体所特有，将属于上面所描述的范围。对于最优氧的反应，最后反应卷应之间 10-20 微升。有小的变化，在反应协议中，取决于基因组的分子形式。例如，线性基因组分子要求附加的组件，在反应中，由 recBCD 酶粗提物中存在抑制线性 DNA 片段的消化。
	<ol><li>完成 &#34; 反应细节 &#34; 节中 表 1 (PhageTXTL_JOVE) 通过输入的反应总数和最后一卷的反应。</li>
		<li>设计实验测定常量元件和变组分的反应。输入的主的组合，到产品的最终反应体积和样本数目不断反应组分总体积之比的体积分数百分比。输入的股票和反应试剂在最终浓度 &#34; 大师混合反应食谱 &#34; PhageTXTL_JOVE 一节。输入的股票和要测试的变量 reagent(s) 最终浓度。 
		注意: 反应组分的卷将自动计算基于主混音音量和每个人的反应的最终数量。</li>
		<li>删除必要量的管 (示 &#34; 管解冻 &#34; PhageTXTL_JOVE.xlsx 节) 原油细胞提取物、 能源缓冲区和氨基酸混合从-20 ° C 或-80 ° C 和冰解冻 
		注: 一次解冻，结合多个整除数的组件一样，（如有必要）。</li>
		<li>分装指示体积的原油提取，33%的最终反应体积，到微量离心管。</li>
		<li>准备按 表 1 的主组合。同质化下的所有组件 &#34; 大师混合反应食谱 &#34; 的旋涡。将适当的卷的每个组件添加到粗提物。</li>
		<li>如果使用噬菌体与线性 DNA 基因组 （例如， T7），添加 1 µ M 的噬菌体 lambda 的 gam 蛋白质反应 11。同质化的解决方案，和在 5 分钟的冰上放置反应的旋涡。这抑制消化的线性 DNA 片段的复杂，recBCD 这是粗提取物中内源性。</li>
		<li>添加按 表 1 的最后一个组件后, 由涡流均质反应。拆分主掺入 n 的微量离心管。 
		注: 每个分裂的体积分数主组合体积百分比的产物和反应的最终数量。例如，90%的分数主组合体积百分比和最终反应体积的 12 µ L，每个拆分量 10.8 微升。</li>
		<li>将可变组件指定的卷添加到主组合阵列 （见 表 1）。添加到每个反应以达到所需的最终反应体积的水。通过涡流均质每个反应。孵化在至少 8 小时或一夜之间 29 ° C 的微量离心管。</li>
	</ol></li> <li>宿主细胞预培养 注: 隔夜前文化是稀释 50: 1，以确保用于滴度实验宿主细胞的中期日志阶段，增加感染效率。中旬日志细胞然后重新集中的 10 倍，存储在冰上。
	<ol><li>使用的无菌针提示，接种 5 毫升磅与 3-5 健康的宿主细胞殖民地文化管。旁边的明火，避免污染工作。</li>
		<li>孵育文化管在振动的孵化器在 37 ° C 和 250 rpm 为 16 h 或一夜之间。细胞可以在第二天到达饱和阶段。</li>
	</ol></li> <li>主机为噬菌体效价细胞文化和样品制备 <ol><li>免除 50 mL LB 到 500 毫升锥形瓶和盖用铝箔。温暖在 37 ° C，20 分钟瓶</li>
		<li>分装 1 毫升的宿主细胞隔夜前文化融入在 37 ° C 的 3-4 小时，摇晃在 250 rpm 预热磅孵育。</li>
		<li>离心机在 5,000 x g.10 分钟 50 毫升宿主细胞培养丢弃磅</li>
		<li>重悬颗粒与冷 (4 ° C) 5 毫升磅和保持上冰。</li>
		<li>一小时后才开始滴度实验，孵育培养板在 37 ° C. 
		注: 每个独特的噬菌体反应 （和相应的稀释倍数） 将利用两个板块。此外，孵育 4 板实验控件 （见 图 1 为代表的阳性和阴性对照）。</li>
		<li>准备 100 毫升的顶级琼脂 （步骤 2.11）。</li>
	</ol></li> <li>准备与磅解决方案的无细胞反应连续稀释 2.12 节中所述。</li>
	<li>噬菌体效价 
	注意: 开始到板后培养皿孵化为至少 1 h 和顶级琼脂是在 45 ° C 水浴为 15-20 分钟后高压灭菌器去除。
	<ol><li>效价最后无细胞反应 LB 解决方案中第 2 款所述步骤 2.14 2.16。</li>
	</ol>
	</li>
</ol>

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作者声明以下竞争金融申请书: Noireaux 实验室获得研究经费从 MYcroarray，MYtxtl 无细胞蛋白质表达工具包的分销商。

后陈等人的技术14的次贷危机，关键的一步被达到确定二重感染的适当条件时。最严密控制主机应变能力，能够承受二重感染的参数通常是感染噬菌体的初始浓度。宿主细胞必须在之前初次感染极少量的噬菌体对数生长阶段。最终，噬菌体也会达到对数增长，目标是以允许噬菌体数字迅速超越细胞计数，允许为每个宿主细胞噬菌体的多个副本。这迫使细胞进入准稳定状态，它将不经过溶解。如果没有达到这种状态，细胞进行级联，汇合的裂解，并释放他们的病毒负荷。如果不能使用上述初始病毒浓度达到二重感染，降低感染的初始多样性进行后续尝试之前的十倍。如果宿主细胞做溶解在收获前和二重感染不能达成，立即着手于一个单独的净化过程。添加头一至 5 微克/毫升和 500 µ L 氯仿3完成所有细胞裂解。继续漩涡额外的 5 分钟，然后用离心机在拥有 10 万 x g 要移除细胞碎片。醒酒，离心上的清液在 75,000 x g 1-2 小时，颗粒噬菌体和重悬一夜之间在 4 ° C 1 毫升总 1 x TM 缓冲区中无晃动。测试的是二重感染使用的细胞体积小，时，能够衡量如何关闭细胞对裂解的他们如何快速溶解时暴露于添加氯仿。如果细胞裂解和解决方案几乎立即澄清，细胞壁是非常不稳定，应立即收获细胞，防止大规模梯级裂解。如果测试溶解在 1-3 分钟内，更大的卷可能会继续 superinfect 达 2 小时，根据大肠杆菌中使用局部应变。如果单元格不做溶解在 5 分钟内，噬菌体生长不赶上了大肠杆菌生长和主要孵化应继续。在 30-60 分钟再次执行测试。
当净化准备的蔗糖梯度，噬菌体噬菌体将显示为三分之一至三分之二的下管方式很厚，珠光的乐队。假设一个相对成功的裂解和细胞残纯化步骤，可能会有其他，轻带但没有相似的密度或大小。大、 不透明带包含噬菌体，而是用无菌注射器和钝器套管从解决方案中移除。
开始第三部分议定书 》，无细胞反应和噬菌体效价时，最好使用新鲜培养板 （少于一周龄） 为宿主菌板 （从步骤 3.5），也用于滴度分析 （从步骤 3.21） 的培养板。这将促进宿主细菌细胞和噬菌体效价高感染效率增长走强。噬菌体培养板必须预先潜伏在 37 ° C 的最低 1 h 开始议定书 》 的噬菌体效价部分之前。没有预孵化的噬菌体培养板，顶部琼脂溶液 （含合成噬菌体与宿主细胞培养） 将不均匀分散文化板的表面。相反，顶部琼脂溶液均匀将巩固在表面 (图 2b)。这增加的可能性多个噬菌体定位在相同的地点，这可能会导致低估的噬菌体产量的无细胞的反应。
每个斑块表示一个合成噬菌体侵染宿主细胞，在宿主细胞内复制和宿主细胞的溶胞的事件。斑块的规模增长孵化期间从子代噬菌体感染的邻居宿主细胞从最初的感染事件。如发现有无斑块，它很可能在议定书第 4.6 节的稀释因素是过高导致任何合成噬菌体感染宿主细胞的可能性很低。若要纠正此问题，减小稀释因子，由 3-4 个数量级 （或必要时） 并重复滴度实验。相反，稀释因素可能是过高导致斑块跨越文化板块的整个表面，使它不可能指望噬菌体产量。在这种情况下，增加稀释因子，由 3-4 个数量级 （或必要时）。精心描绘的噬菌体，将已知无细胞反应收率，每个反应点只有一个稀释因素将是必要滴度实验。描述新的噬菌体，时最好包括 2-3 不同稀释因素每个反应点，确保稀释之一将产生一个可数的牌匾 （见图 3为可数名词和不可数的斑块数目之间的差异上一盘）。
如果没有斑块修改噬菌体效价的稀释因素后观察到的这可能表明，无细胞的反应是不成功，未发生基因表达。为此可能的解释是试剂/噬菌体 DNA 均化无细胞反应的旋涡结构损伤。若要纠正此问题，重复反应和均质化由轻轻敲击反应管五至七倍的一根手指，或慢慢搅拌反应与吸管五至七倍。
无细胞反应系统起源于大肠杆菌和内在包含大肠杆菌RNA 聚合酶和西格玛的主要因素，西格玛 70，相结合，形成酶必须认识到的主要发起人的共识大肠杆菌基因序列。此外，无细胞反应系统可以通过外部提供下西格玛 70 的启动子共识序列六个其他大肠杆菌西格玛因子基因复述整个西格玛因子转录计划。这允许合成所有噬菌体的基因组都与大肠杆菌转录和翻译机器兼容的能力。这留下了噬菌体不能进行研究或合成与无细胞反应系统利用在这里的一个子集。
无细胞转录翻译平台允许令人印象深刻的级别的控制和灵活性的反应条件相比，体内的研究方法。我们可以细调许多生化和遗传元件的噬菌体反应和直接观察的影响在不到 24 小时，为分子拥挤图 4所示。分子 crowders，如挂钩，对影响自组装高分子配合物已经从理论上描述和证明体外18，，1920。分子的拥挤是熵驱动增加了缔合常数的大型生物大分子结构的机制。无细胞的方法使研究者能够探索复杂的生物学过程，像生物物理学的自组装的模型系统，如噬菌体，而体内工作的内在限制。
这个平台还允许在应用领域中的噬菌体的效用的调查。噬菌体基因组组件可以改变用途和快速测试的无细胞反应体系目标纳入新型纳米结构的结果中。噬菌体可以进行修改以增加宿主细胞感染，会促进作为替代抗生素耐药细菌的噬菌体疗法效用的选择性。

在过去的十年，无细胞表达技术工程化到地址小说在新兴的多学科研究领域相关的合成和定量生物学中的应用。最初用于表达蛋白独立的一个活的有机体，新无细胞 TXTL 系统已为这两个基础和应用科学1，2，大大扩大这一技术的范围。新一代的 TXTL 平台已被设计成用户友好，效率更高 （达 2 毫克/毫升的蛋白质合成的批处理模式3），更多功能转录4，一级和模块化，以方便地集成小说自然或扩展现有的生物系统5，6的合成功能。尤其是，无细胞 TXTL 系统已成为方便快速原型的遗传程序，例如调控元件或小基因电路7，8，9，通过减少设计-生成-测试循环到几天。引人注目的是，新的 TXTL 系统也是能够处理大型节目如体内大肠杆菌噬菌10，11，强展示足够的性能支持活跃基因组重组完成合成的 DNADNA 编码生活实体。
TXTL 系统目前相比传统体外建设性的生化分析中的很多技术优势。无细胞 TXTL 链接到最终的产品在减少和开放的环境中，而不是复杂的生活细胞的细胞质基因表达的过程。TXTL 利用 DNA 生化系统体外，重建现代 DNA 装配技术，是负担得起和快除了不需要讲究蛋白质纯化步骤。无细胞的表达提供了大部分的生化反应，从而使分子间的相互作用12深夹层中的组件直接访问。TXTL 反应，人可以改变意愿，几乎是不可能在一个活细胞的生物化学和生物物理设置。鉴于这些优势和最近有所改善，TXTL 技术变得越来越流行作为合成和定量生物学替代平台。虽然快速增长的研究团体使用 TXTL 和 TXTL 正成为标准技术在生物工程中的，至关重要的是要了解如何使用这种平台，发展与执行 TXTL 反应，以及相关的适当做法结果的解释。
在这篇文章中，我们描述了如何使用所有的大肠杆菌TXTL 系统在一锅反应合成噬菌体从其基因组11、 如 MS2 (RNA，3.4 kb)，ΦΧ174 (ssDNA，5.4 kb)，和 T7 (双链 Dna，40 kb)。我们展示如何噬菌体大量合成反应 （镁和钾浓度） 的生化设置的一些变化。分子的拥挤，通过 PEG 范围 8000 浓度，效仿戏剧性影响噬菌体合成了几个数量级。实现的这类大型的生化系统中单试管反应，同时概括的转录，翻译，过程和自组装，是有趣的处理有关的生物学和生物物理学的基本问题。10 (基因调控、 自组装)，以及为开发应用程序，如重新调整用途噬菌体的功能，打造新的纳米结构13。除了实用于 TXTL，我们通过空斑法测定提供噬菌体扩增，基因组提取和纯化，和噬菌体量化的方法。这手稿 》 中提出的方法是适当的人员使用大肠杆菌提取基于无细胞系统和感兴趣的噬菌体。
议定书 》 提出了这项工作可以概括如下: 1） 噬菌体扩增 (一天 1: 准备接种细胞，第 2 天: 单斑块、 多个噬菌体生长和浓度和第 3 天: 纯化噬菌体)，2) 双链基因组 DNA 提取（苯酚/氯仿萃取），和 3) 无细胞噬菌体反应和效价实验 (一天 1: 板宿主细胞并使琼脂平板，第 2 天: 无细胞反应和宿主细胞前文化，和一天 3: 宿主细胞文化和噬菌体效价)。

<table width="899">
	<colgroup>
		<col style="width:162pt" width="216" />
		<col style="width:168pt" width="224" />
		<col style="width:219pt" width="292" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:162pt;height:15.75pt" width="216">Ultracentrifugation tubes</td>
			<td class="xl21" style="width:168pt" width="224">Beckman Coulter</td>
			<td class="xl21" style="width:219pt" width="292">344057</td>
			<td class="xl20" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Conical tubes</td>
			<td class="xl22" style="width:168pt" width="224">Falcon</td>
			<td class="xl21">352070</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Gradient maker</td>
			<td class="xl21">BioComp Gradient Master</td>
			<td class="xl22" style="width:219pt" width="292">see Anal Biochem. 1985 Jul;148(1):254-9.</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Syringe and Blunt Cannula</td>
			<td class="xl21">Monoject</td>
			<td class="xl21">8881513918 and 888202017</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Wide-bore pipette tips</td>
			<td class="xl21">Fischerbrand</td>
			<td class="xl21">02-707-134</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Plaque counter</td>
			<td class="xl21">New Brunswik Scientific</td>
			<td class="xl21">Colony Counter Model C-110</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Culture tubes</td>
			<td class="xl22" style="width:168pt" width="224">Fischerbrand</td>
			<td class="xl21">14-961-33</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Cell-free system</td>
			<td class="xl22" style="width:168pt" width="224">Mycroarray Inc</td>
			<td class="xl22" style="width:219pt" width="292">Mytxtl</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">BioComp Gradient Master</td>
			<td class="xl17" style="width:168pt" width="224">BioComp Instruments</td>
			<td class="xl17" style="width:219pt" width="292">Model 105ME</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">LB agar plate recipe</td>
			<td class="xl20"></td>
			<td class="xl20"></td>
			<td class="xl24" style="width:512pt" width="683">25 g/L Luria-Bertani medium (LB Broth, Miller - Fisher BioReagents product number BP1426) and 15 g/L Bacto-Agar solid (Brenton, Dickenson and Company - product number 214010).</td>
		</tr>
	</tbody>
</table>

synthesis of infectious bacteriophages in an e. coli-based cell-free expression system

新一代的无细胞转录翻译 (TXTL) 系统，工程有更大的灵活性和模块化，提供新颖的功能来执行基础和应用科学中试管反应。在过去的十年，无细胞 TXTL 已成为广泛的新颖的多学科研究领域相关的定量和合成生物学强大技术。新的 TXTL 平台是特别有用的构建和审问生化系统通过合成或天然基因电路的执行。在体外TXTL 已被证明的高度概括，方便快速原型调控元件和生物网络，以及分子自组装机制发现生命系统中。在这篇文章中，我们描述如何传染性噬菌体，如 MS2 (RNA)，ΦΧ174 (ssDNA)，和 T7 (dsDNA)，完全由他们的基因组中使用所有的大肠埃希氏大肠杆菌、 无细胞 TXTL 系统的一锅反应合成。合成三个体内大肠杆菌噬菌被量化使用空斑法测定。我们展示如何合成噬菌体产量取决于反应的生化设置。分子的拥挤，通过控制浓度的 PEG 8000，效仿的订单的大小影响合成噬菌体的量。我们还描述如何放大噬菌体和如何净化它们的基因组。组协议和结果提出了这项工作应该是多学科研究人员感兴趣的在无细胞合成生物学与生物工程学院。

我们显示四个标志性的成果。在图 1中，我们提出一套的阴性对照，以确保无细胞 TXTL 系统和噬菌体 DNA 股票不沾染生活大肠杆菌细胞。我们验证无细胞 TXTL 系统是免费的完整大肠杆菌细胞由电镀无效的基因组 DNA （图 1A和图 1B） 两个非孵化和孵育反应液的污染。如果无细胞 TXTL 系统被污染了大肠杆菌细胞，将板上观察生长。此外，我们的无细胞的反应，并因此没有任何可能的污染物细胞，通过验证负责的噬菌体合成电镀非孵化、 孵育反应，有的噬菌体基因组 （图 1C和图 1D)。与非孵化反应 (图 1C) 板显示零斑块而与孵育反应 (图 1D) 板显示斑块，该值指示单元格免费 TXTL 系统负责噬菌体合成。
在图 2中，我们比较均匀与非均匀空斑法测定。图 2B说明跨文化板块，这是比较均匀的斑块密度板在图 2A的次优的斑块密度梯度。源本观察结果是从主组合顶级的琼脂、 样品和主机细菌细胞，当在进行实验的噬菌体效价部分配发到培养板的快速冷却。为了避免噬菌体斑块不均匀扩散，确保培养板开始噬菌体效价实验之前，到 37 ° C 的平衡。
关键的一步正确计算噬菌体是稀噬菌体溶液足以获得之间 50 和 200 斑块每板 (图 3)。低于这个范围，很难确定真实的数据，并且高于此范围内，斑块重叠，防止准确计数。在 TXTL 中合成的噬菌体的数量取决于生化的设置，如浓度的盐 （钾、 镁）、 分子 crowders，和基因组。在图 4中，我们展示噬菌体 T7 和 ΦΧ174 测试几个的例子。复制它的 DNA，如 T7，噬菌体为我们观察到的噬菌体合成中的无细胞的反应效率高: 每个基因组分子反应合成了三个传染性噬菌体。谁都可以预料不会复制它的 DNA，如 ΦΧ174 噬菌体反应低效率: 合成传染性噬菌体基因组分子的比率是 1:5。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56144/56144fig1.jpg"> 
图 1:斑块检测控制及使用无细胞反应 (CFR) 系统的成功传染性噬菌体生产。(A) 没有基因组被添加和 CFR 没有孵化 （作为孵化时间 0 分钟） 进行。反应了然后镀无主机大肠杆菌。结果表明没有可行的宿主细胞污染的 CFR。(B) 没有基因组被添加和 CFR 是孵化 12 h 在 29 ° C 和镀无主机大肠杆菌。结果表明没有可行的宿主细胞污染的 CFR，甚至后孵化。(C) 1 毫微米基因组附带没有孵化的 CFR （作为孵化时间 0 分钟） 和镀与大肠杆菌的主机。结果表明基因组解决方案无噬菌体污染。(D) 1 毫微米基因组包括和 CFR 孵化 12 h 为 29 ° c。和镀与大肠杆菌的主机。结果表明在 CFR 成功传染性噬菌体复制。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56144/56144fig2.jpg"> 
图 2: 均匀与非均匀扩散的噬菌体效价试验期间反应。噬菌体效价进行培养板不平衡到 37 ° c 的温度可能影响跨文化板是噬菌体反应的非均匀传播。一个好的结果，显示均匀扩散的噬菌体反应所示 (A)，在那里斑块会均匀分布在板的表面。次优的结果所示 (B)，在那里斑块都集中在培养板的左下角部分。这可能发生与顶部-琼脂，噬菌体反应和宿主细胞混合配发到培养板，不是在一个温度 37 ° c。主混合绝大多数凝固在左下角区域，和均匀的传播是不可能。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56144/56144fig3.jpg"> 
图 3: 设置适当稀释因子为无细胞噬菌体反应效价。(A) 此板显示合理可数个斑块。用于实验的效价部分稀释倍数稀释产量的噬菌体反应不够，在板上留下数个斑块。相反在 (B) 稀释因子对合成噬菌体，产量太低，需要进行调整。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56144/56144fig4.jpg"> 
图 4:噬菌体的无细胞合成表征ΦΧ174 和 T7。(A) 数目合成的 ΦΧ174 和 T7 噬菌体 (PFU/mL: plaque 成型机组每毫升） 作为一个函数的 PEG 8000 浓度在无细胞反应中，测量后 16 小时的潜伏期在 29 ° c。(B) 合成的 ΦΧ174 数和数的 T7 噬菌体中的无细胞的反应，镁谷氨酸浓度的函数测量后 16 小时的潜伏期在 29 ° c。(C) 数目合成的 ΦΧ174 噬菌体作为函数的 ΦΧ174 DNA 基因组浓度在无细胞反应中，测量后 16 小时的潜伏期在 29 ° c。(D) 数目合成 T7 噬菌体 T7 DNA 基因组浓度在无细胞反应中，一个函数作为衡量后 16 小时的潜伏期在 29 ° c。显示的所有误差线都是标准偏差的三个重复试验。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144fig4large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Table 1" src="/files/ftp_upload/56144/56144table1.jpg"> 
表 1: 反应组成。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144table1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>

Watch this Scientific Journal Video about Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System at JoVE.com

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

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

新一代的无细胞的转录翻译平台被设计在生化系统体外基因电路执行构建。在这篇文章中，我们描述如何从其基因组，使用所有的大肠杆菌细胞免费 TXTL 系统合成噬菌体 MS2、 ΦΧ174 和 T7，等。

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

synthesis-infectious-bacteriophages-an-e-coli-based-cell-free

Nanyang Technological University

Research

JoVE Journal

Bioengineering

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

14.5K 次观看.  University of Minnesota. 新一代的无细胞的转录翻译平台被设计在生化系统体外基因电路执行构建。在这篇文章中，我们描述如何从其基因组，使用所有的大肠杆菌细胞免费 TXTL 系统合成噬菌体 MS2、 ΦΧ174 和 T7，等。

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

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

科研

生物工程

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

快速生长的纳氏弧菌的无细胞蛋白表达

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

测量组织直肠炎和莱蒂克药物血栓分析功效的体外系统

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...

OnePot PURE Cell-Free System

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

一锅纯无细胞系统

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

使用无细胞系统快速表征遗传部分

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.