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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H., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+dynamics+of+protein+synthesis+in+a+cell-free+system.">Coarse-grained dynamics of protein synthesis in a cell-free system.</a> Phys Rev Lett. 106 (4), 048104 (2011).</li><li>Daube, S. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding:+biochemical,+biophysical,+and+physiological+consequences.">Macromolecular crowding: biochemical, biophysical, and physiological consequences.</a> Annu Rev Biophys Biomol Struct. 22, 27-65 (1993).</li></ol>

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?...

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><tbody><tr><td>Ultracentrifugation tubes</td><td>Beckman Coulter</td><td>344057</td><td></td></tr><tr><td>Conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Gradient maker</td><td>BioComp Gradient Master</td><td>see Anal Biochem. 1985 Jul;148(1):254-9.</td><td></td></tr><tr><td>Syringe and Blunt Cannula</td><td>Monoject</td><td>8881513918 and 888202017</td><td></td></tr><tr><td>Wide-bore pipette tips</td><td>Fischerbrand</td><td>02-707-134</td><td></td></tr><tr><td>Plaque counter</td><td>New Brunswik Scientific</td><td>Colony Counter Model C-110</td><td></td></tr><tr><td>Culture tubes</td><td>Fischerbrand</td><td>14-961-33</td><td></td></tr><tr><td>Cell-free system</td><td>Mycroarray Inc</td><td>Mytxtl</td><td></td></tr><tr><td>BioComp Gradient Master</td><td>BioComp Instruments</td><td>Model 105ME</td><td></td></tr><tr><td>LB agar plate recipe</td><td></td><td></td><td>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?...

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 ...

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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

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

科研

免疫与感染

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

生物学

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

化学

<meta charset="utf8"></head><body>用于微区室化合成细胞的 CFPS 方案

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

<meta charset="utf8"></head><body>作者聚焦：优化 CFPS 系统以进行合成细胞构建

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

用于构建合成细胞的无细胞蛋白质合成系统

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

生物化学

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

生物工程

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

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.

使用无细胞自动诱导工作流程在24小时内从细胞到无细胞蛋白质合成

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

传染性噬菌体大肠杆菌-合成基于无细胞表达系统

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在细胞命运

大型插入环境基因组文库生产

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

用于构建合成细胞的无细胞蛋白质合成系统

用于构建合成细胞的无细胞蛋白质合成系统

用于构建合成细胞的无细胞蛋白质合成系统

基于多步法链位移光刻的基因功能表面固定化研究

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

使用无细胞自动诱导工作流程在24小时内从细胞到无细胞蛋白质合成

协议用于实现