我们感谢Jongmin金，丹西耶戈尔 - 加斯金斯，阿努Thubagere，和杨诺为协助精简协议，克莱尔和陈李巴克利在项目的早期阶段帮助。这种材料是基于由美国国防高级研究计划局（DARPA / MTO）的部分资助生活铸造程序，合同编号HR0011-12-C-0065（DARPA / CMO.ZZS也支持由加州大学洛杉矶分校/加州理工学院医学科学家的工作培训项目奖学金和由国防部，科学研究，国防科学与工程研究生（NDSEG）奖学金，32 CFR 168A的空军办公室。本文件所载的观点和结论是那些作者的，不应该被解释为代表正式的政策，不论是明示的国防高级研究计划局和美国政府或暗示的保证。

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作者宣称，他们有没有竞争的财务权益。

内源性大肠杆菌根据这里所描述TX-TL无细胞表达系统是一个易于运行三级管反应，可以采取少于八个小时，从成立到数据收集。创建的所有试剂的过程中，需要五天的时间总量（与仅一天显著劳动力需求），但产生的粗提取物为3000和反应缓冲液，使试剂为10,000的反应（ 图1）。此外，粗提物和缓冲，使试剂可保持稳定至少1年，在-80°C，使一个准备的多种用途4。在$ 0.11每10μl反应体系（0.26美元包括劳动力），成本比同类低98％商业系统（ 图2）。 然而，也有一些未解决的限制，该系统。每个细胞粗提取物制剂的端效率可以根据用户的能力和对环境条件的变化，虽然吨YPICAL产量变化是在5-10％之间（ 图4b）。因此，批与批之间的变化在两个端点表达和表达动力学是可以预料的。这些变化可能会保持，直到提取物充分的特点，或直至提取物创作是完全自动化的。如果无细胞表达系统用于进行敏感的定量实验中，最好是用相同批次的细胞粗提液上运行的所有实验。从一个单一的细胞粗提液批次，约3000反应中，产率应该是足够的典型实验课程。虽然我们怀疑的变化可以通过扩大和自动化的程序进行删除，这样的尝试会涉及到大量的资源投入。 另外，虽然终点的表达水平是相当容易确定，更多的工作需要在理解动力学固有的无细胞系统来完成。它是已知的，这两个资源competition和资源限制会影响表达动态。例如，有限的内生西格玛70可导致饱和制度与增加DNA模板制备表达谱类似于核苷酸或氨基酸的耗竭。9,27然而，动力学没有被完全理解，利用该系统。对于收益率的纯增加，优化可以通过机器学习的方法来完成。资源竞争和限制28问题可以通过数学模型实验数据处理验证。 这里提出的协议是为BL21-Rosetta2菌株进行了优化，但推广到其他E。大肠杆菌菌株。修改在BL21-Rosetta2，如除去编码基因Lon蛋白酶和另外的基因编码稀有tRNA的的，允许的最大的蛋白质生产。我们已经尝试了协议，与其他两个菌株提取物-只有BL21和BL21一个淘汰赛的trxA安ð发现少了50％蛋白产量。我们假设单产使用其他菌株时同样减少。在参数，如开关的2×YT培养基为LB和其他丰富的肉汤等变化，导致减少蛋白质产量。 利用内源性和外源性转录-翻译机械和调控机制的无细胞表达系统中有两种蛋白质和代谢物的表达和在合成生物学广泛的应用，而是被限制在T7调节电路3,29，人们可以设想产生复杂的生物分子在使用本机E的 ​​组合使用者可控制的设定大肠杆菌的启动子和外源转录和调控机制。无细胞分裂和代谢的局限性，可变性在合成电路如repressilator或在代谢工程化途径，如那些生产青蒿素可以减少或更好的理解。30,31我们甲肝e一起使用的这些优点来实现基因开关，以及理解σ因子封存9,32这样的技术还可以形成的“最小”或“人造”的细胞骨干-小的，良好表征的和自足的体现单位提取物。33,34 最终，我们预期这种内源性的无细胞表达系统的直接用途为合成生物学原型环境。绰号“TX-TL生物分子实验电路板”的无细胞表达系统提供了一个可控制的环境下，合成电路最终目的地是在体内表达可以进行几轮原型-测试的基础质粒，线性的，或化学原料合成的DNA周期，其次通过分析和快速修改。原型回合可以通过目前正在开发预测的数学模型来辅助。通过消除克隆和体内操纵非最终电路，我们预计工程师neering周期时间将减少，而不是当前周的标准为1-3天。

A correction to Figure 5's legend has been made for the article <a href="http://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free">Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology</a>. Method 1 and method 2 have been switched.Z
The figure legend was update from:
Figure 5. Effects of DNA solution on expression efficiency. a) Comparison of two different purification methods for processing plasmids. 1 nM of pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is prepared using only a QiaPrep Spin Miniprep Kit (Purification method 1) or post-processed with a QiaQuick PCR purification kit (Purification method 2).
to
Figure 5. Effects of DNA solution on expression efficiency. a) Comparison of two different purification methods for processing plasmids. 1
nM of pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is prepared using only a QiaPrep Spin Miniprep Kit (Purification method 2) or post-processed
with a QiaQuick PCR purification kit (Purification method 1).

A correction to Figure 5's legend has been made for the article Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Method 1 and method 2 have been switched.

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

无细胞表达技术始于20世纪50年代作为纯粹的平移，推动年后用T7噬菌体的DNA为包括转录-翻译机制。11,12从那时起，已经作出许多努力来优化创作的细胞粗提液（或E大肠杆菌 S30提取物）。13,14这些优化包括通过ATP的再生或应变修改延长无细胞蛋白质合成，减少协议的时间和成本。15-17另类无细胞表达系统存在的替代原油的使用再造成分细胞提取物的表达。5两个细胞粗提液和重建方法已被开发用于商业用途。 与合成生物学的出现，为充分表征的平台的需要增加，以测试和快速工程化生物模块和电路。18,19此平台必须是多功能，良好的特点，简单的​​操作，并专注于用户提供的组件。尽管正在开发一个半世纪前，基于大肠杆菌无细胞系统大肠杆菌本质上共享这些要求，因为它们是一个简化的细胞过程的体外表示无生长和代谢的复杂性。此外，所有来自于大肠杆菌体内工作的基础知识大肠杆菌容易适用于E。大肠杆菌无细胞系统。 虽然无细胞表达系统可以具有应用在合成生物学，迄今大多数无细胞表达系统的目标一直是蛋白质和代谢物的产率最大化。这是通过使用由T7启动子驱动序列的T7噬菌体的转录来实现的。20虽然表达是高效且健壮的，这些系统成为一个高度专业化的目的。细胞的调节方法是有限的，目标DNA模板必须是重新设计，包括T7启动子，和某些序列，如核糖体复合物不能被转录和组装。21,22现有的无细胞表达系统是无法维持高产量，同时保持内源性调控机制，对于合成生物学所必需的灵活性。 我们已经开发出一种内源性E。它保留蛋白的表达由先前的系统中表现出的效率，而且通过允许增加额外的多功能性大肠杆菌无细胞表达系统中表达和基于内源性和外源性（T7或其他）的机制调节。这里所描述的协议最初是根据木川等人（2004）和Liu 等人 （2005年），但具有显著修改。它利用的Mg-和K-谷氨酸以上的Mg-和K-醋酸为提高效率，消除2 -巯基乙醇，并用珠打浆机裂解细胞。17,23,24微珠打浆被选择过均质化，基于压力的方法，或超声处理，由于其较低的成本和可比较的收率，以竞争系统23 3 -磷酸甘油酸（3-PGA）作为能量源，因为它被发现时相比，磷酸肌酸，得到优良的蛋白质的产率和磷酸烯醇丙酮酸。4,25我们的系统可以使用一个sigma70基于启动子与λ噬菌体运营商或T7-启动子驱动，从其他类似的商业系统的产量产生高达报告蛋白的0.75毫克/毫升。4,6五天须出示所有必需的试剂（ 图1）。此外，它提供了一个降低98％的成本相比同类商业无细胞系统-材料成本是$ 0.11每10μl反应体系，其中上升0.26美元与劳动包括：（ 图2）。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>2xYT</td>
 <td>MP biomedicals</td>
 <td>3012-032</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3-PGA</td>
 <td>Sigma-Aldrich</td>
 <td>P8877</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A8937</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bacto-agar</td>
 <td>BD Diagnostics</td>
 <td>214010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bead-beating tubes (polypropylene microvials)</td>
 <td>BioSpec</td>
 <td>522S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Beads, 0.1mm dia.</td>
 <td>BioSpec</td>
 <td>11079101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BL21 Rosetta 2 E. coli strain</td>
 <td>Novagen</td>
 <td>71402</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bradford BSA Protein Assay Kit</td>
 <td>Bio-rad</td>
 <td>500-0201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>cAMP</td>
 <td>Sigma-Aldrich</td>
 <td>A9501</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C1919</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CoA</td>
 <td>Sigma-Aldrich</td>
 <td>C4282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CTP</td>
 <td>USB</td>
 <td>14121</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cuvettes, 1.5ml</td>
 <td>Fisher</td>
 <td>14-955-127</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Sigma-Aldrich</td>
 <td>D0632</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Folinic acid</td>
 <td>Sigma-Aldrich</td>
 <td>F7878</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GTP</td>
 <td>USB</td>
 <td>16800</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H6147</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>K-glutamate</td>
 <td>Sigma-Aldrich</td>
 <td>G1149</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mg-glutamate</td>
 <td>Sigma-Aldrich</td>
 <td>49605</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro Bio-Spin Chromatography Columns</td>
 <td>Bio-Rad</td>
 <td>732-6204</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NAD</td>
 <td>Sigma-Aldrich</td>
 <td>N6522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nunc 384-well optical bottom plates</td>
 <td>Thermo-Scientific</td>
 <td>142761</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nunc sealing tape</td>
 <td>Thermo-Scientific</td>
 <td>232701</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PEG-8000</td>
 <td>Promega</td>
 <td>V3011</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium phosphate dibasic solution</td>
 <td>Sigma-Aldrich</td>
 <td>P8584</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium phosphate monobasic solution</td>
 <td>Sigma-Aldrich</td>
 <td>P8709</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RTS Amino Acid Sampler</td>
 <td>5 Prime</td>
 <td>2401530</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Slide-A-Lyzer Dialysis Cassettes, 10k MWCO (Kit)</td>
 <td>Thermo-Scientific</td>
 <td>66382</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spermidine</td>
 <td>Sigma-Aldrich</td>
 <td>85558</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris base</td>
 <td>Fischer</td>
 <td>BP1521</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>tRNA (from E. coli)</td>
 <td>Roche Applied Science</td>
 <td>MRE600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>UTP</td>
 <td>USB</td>
 <td>23160</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1L Centrifuge Bottle</td>
 <td>Beckman-Coulter</td>
 <td>A98813</td>
 <td>This is specific for Avanti J-series; obtain equivalent size for centrifuge in use.</td>
 </tr>
 <tr>
 <td>4L Erlenmeyer Flask</td>
 <td>Kimble Chase</td>
 <td>26500-4000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Avanti J-26XP Centrifuge</td>
 <td>Beckman-Coulter</td>
 <td>393127</td>
 <td>Or 1L-capable centrifuge equivalent.</td>
 </tr>
 <tr>
 <td>Forma 480 Orbital Shaker</td>
 <td>Thermo Scientific</td>
 <td>480</td>
 <td>Or chest-size 6x4L shaker equivalent.</td>
 </tr>
 <tr>
 <td>JLA-8.1000 Rotor</td>
 <td>Beckman-Coulter</td>
 <td>363688</td>
 <td>Or 1L-capable, 5000 x g rotor equivalent for centrifuge.</td>
 </tr>
 <tr>
 <td>Mini-Beadbeater-1</td>
 <td>BioSpec</td>
 <td>3110BX</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
Supplemental Material 1. Recipes for Items. 
Chloramphenicol, 34 mg/ml: Prepare 0.51 g chloramphenicol and add ethanol to 15 ml. Filter sterilize (0.22 &mu;M), aliquot to 1 ml tubes, store at -20 &deg;C for later use. 
2xYT+P+Cm agar plate: Prepare 1.24 g 2xYT, 1.6 ml potassium phosphate dibasic solution @ 1 M, 0.88 ml potassium phosphate monobasic solution @ 1 M, 0.6 g agar, and water to 40 ml. Autoclave. Let cool to 50 &deg;C and add 40 &mu;l Cm. Aliquot 25 ml into a 100x15 mm petri dish, and let cool for an hour. 
2xYT+P media: Prepare 124 g 2xYT, 160 ml potassium phosphate dibasic solution @1 M, 88 ml potassium phosphate monobasic solution @ 1 M, and water to 4 L. Aliquot out into 2x1.88 L and 0.24 L. Autoclave. 
Tris base, 2 M: Prepare 60.57 g Tris base and water to 250 ml. Sterilize, store at RT for later use. 
DTT, 1 M: Prepare 2.31 g DTT and water to 15 ml. Filter sterilize (0.22 &mu;M), aliquot to 1 ml tubes, store at -20 &deg;C for later use. 
S30A buffer: Prepare 10.88 g Mg-glutamate and 24.39 g K-glutamate, 50 ml Tris at 2M, acetic acid (to pH 7.7), and water to 2 L. Autoclave, store at 4 &deg;C, add 4 ml 1 M DTT before use. 
S30B buffer: Prepare 10.88 g Mg-glutamate and 24.39 g K-glutamate, Tris at 2 M (to pH 8.2), and water to 2 L. Autoclave, store at 4 &deg;C, add 2 ml 1 M DTT before use. 
HEPES: Prepare 1.91 g HEPES (MW 238.21), KOH (to pH 8), and water to 4 ml. 
tRNA: Prepare 30 mg of tRNA and water to 600 &mu;l. 
CoA: Prepare 30 mg of CoA (MW 767.53) and water to 600 &mu;l. 
NAD: Add 34.83 mg of NAD (MW 663.43), Tris at 2 M (to pH 7.5-8), and water to 300 &mu;l. (Add 27 &mu;l of Tris at 2 M to bring the solution to pH 7.5-8). 
cAMP: Add 42.80 mg of cAMP (MW 329.22), Tris at 2 M (to pH 8), and water to 200 &mu;l. (Add 73 &mu;l of Tris at 2 M to bring the solution to pH 8). 
Folinic Acid (33.9 mM): To 20 mg of solid folinic acid calcium salt (MW 511.5), add 1.15 ml water. 
Spermidine: Prepare 23.55 &mu;l of spermidine (MW 145.25) and water to 150 &mu;l. Prepare at room temperature after melting briefly at 37 &deg;C. 
3-PGA: Add 1.03 g of 3-PGA (MW 230.02), Tris at 2 M (to pH 7.5), and water to 3.2 ml. (Add 1.73 ml of Tris at 2 M to bring the solution to pH 7.5). 
Nucleotide Mix: Add 145 mg of ATP dipotassium salt dihydrate (MW 619.4), 133 mg of GTP disodium salt (MW 567.14), 79.4 mg of CTP disodium salt dihydrate (MW 563.16), 82.6 mg of UTP trisodium salt dihydrate (MW 586.12), KOH at 15% dilution (to pH 7.5), and water to 1.5 ml. (Add 353 &mu;l of KOH at 15% dilution to bring the solution to pH 7.5). 
Supplemental Material 2. Bradford Assay.
<ol>
<li>Remove Bradford agent from 4 &deg;C and set at room temperature.</li>
<li>Prepare 50 &mu;l BSA Standard at 1 mg/ml and at 0.1 mg/ml.</li>
<li>Prepare 40 &mu;l 20x dilution of extract from step 1.47.</li>
<li>Add 800 &mu;l water to 7 cuvettes. </li>
<li>Prepare standard cuvettes for 0 mg/ml, 1 mg/ml (10 &mu;l 0.1 mg/ml BSA), 2 mg/ml (20 &mu;l 0.1 mg/ml BSA), 4 mg/ml (4 &mu;l 1 mg/ml BSA), 6 mg/ml (6 &mu;l 1 mg/ml BSA).</li>
<li>Prepare experimental cuvettes for 2 &mu;l of sample and 4 &mu;l of sample.</li>
<li>Add 200 &mu;l of Bradford agent to each cuvette and mix well by pipetting. Incubate at room temperature for at least 10 min.</li>
<li>Produce standard curve at OD 595nm using cuvettes from step 6.5. Reject standard curve if r2 &lt; 0.95.</li>
<li> Determine extract concentration at OD 595nm using cuvettes froms tep 6.6.</li>
</ol>
Supplemental Material 3. Buffer calibration spreadsheet. 
<a href="http://www.jove.com/files/ftp_upload/50762/TXTL_e(template)_calibration_JoVE.xlsx" target="_blank">See TXTL_e(template)_calibration_JoVE.xlsx</a>.
 
Supplemental Material 4. Cell-free expression run spreadsheet. 
<a href="http://www.jove.com/files/ftp_upload/50762/TXTL_JoVE.xlsx" target="_blank">See TXTL _JoVE.xlsx</a>.
 </td>
 </tr>
 </tbody>
</table>

protocols for implementing an escherichia coli based tx-tl cell-free expression system for synthetic biology

理想的无细胞表达系统在理论上可以模拟体内细胞的环境中以受控的体外平台1，这是用于表达的蛋白质和基因的电路以控制的方式，以及用于提供原型环境为合成生物学有用的。2,3为了实现后者的目标，即保持无细胞表达系统的内源性大肠杆菌转录转译机制能够更准确地反映体内细胞动力学比那些基于T7 RNA聚合酶转录。我们描述一个高效的内源性大肠杆菌的准备和执行基于大肠杆菌转录-翻译（TX-TL）无细胞可在98％的成本降低到类似的商业系统产生的蛋白质为T7为基础的系统等量表达系统。4,5缓冲器和细胞粗提液的制备方法描述，以及一个执行三级管TX-TL反应。整个协议需要五天的时间准备，并产生足够的材料多达3000单一反应在一种制剂。一旦准备，每个反应根据需要从建立到数据收集和分析8小时。调控和外源性转录到E的机制大肠杆菌 ，如lac /四面体阻遏和T7 RNA聚合酶，可以进行补充。6内源性质，例如mRNA和DNA的降解率，也可以进行调整。7该TX-TL无细胞表达系统已被证明对于大规模的电路组装，探索生物现象，和蛋白质的两个T7和内源性启动子下表达。6,8陪同数学模型可供选择。9,10由此产生的系统在合成生物学作为一个原型环境的独特应用，或“TX- TL生物分子实验电路板。“

我们已经提出了五天的协议，用于基于TX-TL无细胞表达系统中的内源性大肠杆菌的准备。细胞粗提液和缓冲液- -用于创建试剂样品时间轴可以在图1中可以找到。一旦创建，这些都可以保存在-80℃可保存一年。创建试剂后，实验装置和执行可以在不到8小时来完成。 此外，我们优化了TX-TL无细胞表达系统表达的条件。其他用户提供的补充，如缓冲区或DNA溶液，应事先标定的毒性。例如，处理的质粒的不同方法由于盐含量会导致不同的表达。我们还测试了的Tris-氯洗脱缓冲液对反应效率（ 图5）的效果。 的细胞粗提液的校准，参照步骤4.1至4.9的示例中，示出在图4a中。在一般情况下，我们的实验表明，该细胞粗提液是最敏感的Mg-谷氨酸水平，其次是谷氨酸钾水平。为了证明无细胞表达系统，我们构建和测试基于四环素压制一个负反馈回路26（ 图6）。在无细胞表达系统，带或不带ATC相同的电路运行显示deGFP记者八小时后表达的7倍终点的表达变化。虽然这个实验并不需要全球诱导或阻遏，如有必要，他们可以按“主混音准备。”加<img alt="图1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50762/50762fig1highres.jpg" src="/files/ftp_upload/50762/50762fig1.jpg" /> 图1。时间轴细胞粗提液，氨基酸溶液，和能源解决方案的准备。为期五天的timelin<html> e表示该协议的典型执行上面给出，过夜孵育和白天的工作步骤进行了优化。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig2highres.jpg" src="/files/ftp_upload/50762/50762fig2.jpg" /> 图2。成本和竞争的细胞粗提物的表达分析。一）击穿的TX-TL无细胞表达系统的人工和材料的费用。根据试剂的成本，2012年12月，和14元每小时的劳动力成本。 二）TX-TL无细胞表达系统成本与其他商业系统的比较。成本每微升细分，虽然反应体积可能会有所不同每包。C的 TX-TL无细胞表达系统的产量与其他商业系统）的比较。蛋白表达产量由制造商标准确定。/ 50762fig2large.jpg“目标=”_blank“&gt;点击这里查看大图。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig3highres.jpg" src="/files/ftp_upload/50762/50762fig3.jpg" /> 图3。加载中珠跳动管和处理。一）细胞珠液粘度正确的示范。 B）珠跳动管装入前快速桌面离心细胞珠的解决方案将有一个粘度取决于许多因素，包括S30A缓冲区添加量，添加的量珠，和时间花在冰上。离心去除加载过程中积累的泡沫。 三）台式离心后的气泡浮出水面。气泡的大小会有所不同的，他们可以用枪头D为弹出或删除）完全填充珠跳动管封盖之前。形成于珠跳动吨一个弯月形宇部，和盖有足够的覆盖，并造成少量的过满。 五）正确装入过滤装置。这些可重复使用正确与不正确地处理珠跳动管。F）的比较。左边的管子是一个良好的节拍管 - 它设有一个小型和精心描绘的顶层，而且非常透明的上清液。右边的管子是最理想的，基于更大的，朦胧的第二层和朦胧的上清液上。管是最理想的不应该进行额外的处理。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig4highres.jpg" src="/files/ftp_upload/50762/50762fig4.jpg" /> 图4。粗提物制剂特性。一）典型的校准曲线为细胞粗提液。粗提取物进行校准，以获得更多的Mg-谷氨酸盐，谷氨酸钾，和DTT的水平，按该顺序。显示的是端点fluorescencË后8小时，以及蛋白生产的基础上有12分钟的移动平均线的最大速率。基于这些情节，额外的Mg-谷氨酸可接受的范围是4毫米，谷氨酸钾是60-80毫米，和DTT是0-3毫米。需要注意的是每一个粗提物需要进行独立校准的这三个变量。 二）从提取物制剂的变化。编制日期不同的两个粗提物端点荧光显示，误差线是从1在不同的日子三名独立运行的标准偏差。 <a href="https://www.jove.com/files/ftp_upload/50762/50762fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig5highres.jpg" src="/files/ftp_upload/50762/50762fig5.jpg" /> 图5。对影响表达效率的DNA溶液。两种不同的纯化）的比较方法处理的质粒。 1纳米的pbest-OR-OR-PR-UTR1-deGFP-T500时，采用只有一个QIAprep离心微量制备试剂盒（纯化方法1）或后处理，用QIA快速PCR纯化试剂盒制备的（纯化方法2）。示出的是8小时，以及蛋白质产量的基础上在12分钟的移动平均最大比率后端点荧光。误差棒是从1在不同的日子四个独立运行的标准偏差。洗脱缓冲液b）对 （的Tris-Cl计）。不同浓度的Tris-氯进行比较的基础上的1纳米的pbest-OR-OR-PR-UTR1-deGFP-T500的表达的无细胞表达反应。给定浓度在反应的最终浓度的Tris-氯;用洗脱缓冲液是10毫摩尔Tris-CL。误差棒是从1在不同的日子三名独立运行的标准偏差。 <a href="https://www.jove.com/files/ftp_upload/50762/50762fig5large.jpg" target="_blank">点击这里查看大图</a> 。 图6。一个负反馈回路的采样TX-TL运行。 a）样品无细胞反应执行的设置。 “上”与负反馈回路的“关闭”状态，与阳性和阴性对照。 二）负反馈回路的质粒图谱。 三）代表结果的测试。数据反映了实验中a）和b）中，与阴性对照组相减信号。显示在插入基因电路。误差棒是从1在不同的日子三名独立运行的标准偏差。 <a href="https://www.jove.com/files/ftp_upload/50762/50762fig6large.jpg" target="_blank">点击这里查看大图</a> 。 <table border="1"><tbody><tr><td> 名称 </td><td> 浓度 </td><td> 量 </td><td> 消毒 </td><td> 笔记 </td></tr><tr><td>氯霉素（厘米） </td><td> 34毫克/毫升在乙醇中</td><td> 1毫升</td><td>过滤消毒（0.22微米） </td><td>可以保存在-20℃下以备后用较大的体积进行。 </td></tr><tr><td>的2×YT + P +厘米琼脂平板</td><td> 31克/ L的2×YT，40 mM磷酸氢二钾，22 mM磷酸钾一元，34微克/毫升氯霉素</td><td> 1板</td><td>高压灭菌器</td><td></td></tr><tr><td>的2×YT + P媒体</td><td> 31克/ L的2×YT，40 mM磷酸氢二钾，22 mM磷酸钾一元</td><td> 4升</td><td>高压灭菌器</td><td></td></tr></tbody></table> 表1中。试剂的细胞粗提液协议的第1天。 <table border="1"><tbody><tr><td> 名称 </td><td> 浓度 </STRONG&gt; </td><td> 量 </td><td> 消毒 </td><td> 笔记 </td></tr><tr><td> Tris碱</td><td> 2M的</td><td> 250毫升</td><td>过滤消毒（0.22微米）或高压灭菌器</td><td>可在室温下保存。 </td></tr><tr><td> DTT </td><td> 1个月</td><td> 6毫升</td><td>过滤消毒（0.22微米） </td><td>可以制成较大的体积，并存储于-20℃以备后用。 </td></tr><tr><td> S30A缓冲区</td><td> 14毫米的Mg-谷氨酸盐，60mM的K-谷氨酸的50mM Tris，pH值7.7 </td><td> 2升</td><td>高压灭菌器</td><td>至pH值达到7.7，滴定用乙酸。加入DTT至2毫米的最终浓度使用前。贮存于4℃。 </td></tr><tr><td> S30B缓冲</td><td> 14毫米镁-谷氨酸，60毫米的K-谷氨酸，〜5毫米的Tris，pH值8.2 </td><td> 2升</td><td>高压灭菌器</td><td>为了达到pH值为8.2，滴定2M三。加入DTT至1 mM的终浓度使用前。贮存于4℃。 </td></tr></tbody></table> 表2。试剂为细胞粗提液协议的第2天。 <table border="1"><tbody><tr><td></td><td colspan="4"> 鹘 </td></tr><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td></tr><tr><td>空50毫升猎鹰（G） </td><td></td><td></td><td></td><td></td></tr><tr><td> 50毫升猎鹰颗粒（G） </td><td></td><td></td><td></td><td></td></tr><tr><td>颗粒质量（50毫升猎鹰与沉淀-空50毫升猎鹰）（G） </td><td></td><td></td><td></td><td></td></tr><tr><td> S30A缓冲区量增加（颗粒质量* 0.9）（毫升） </td><td></TD&gt; <td></td><td></td><td></td></tr><tr><td>珠总质量增加（颗粒质量* 5.0）（G） </td><td></td><td></td><td></td><td></td></tr></tbody></table> 表3。 S30A缓冲区和珠质量计算器，用于细胞粗提液协议的第3天。 <table border="1"><tbody><tr><td> 名称 </td><td> 浓度 </td><td> 量 </td><td> 消毒 </td><td> 笔记 </td></tr><tr><td> HEPES </td><td> 2 M，pH值8 </td><td> 4毫升</td><td>无</td><td>为了达到pH值为8，滴定用KOH。 </td></tr><tr><td>核苷酸混合物</td><td> 156毫摩尔ATP和GTP，94毫米的CTP和UTP，pH值7.5 </td><td> 1.5毫升</td><td>无</td><td>为了达到pH为7.5，滴定用KOH。 </td></tr><tr><td>氨酰tRNA </td><td> 50毫克/毫升</td><td> 600微升</td><td>无</td><td></td></tr><tr><td>辅酶A </td><td> 65毫米</td><td> 600微升</td><td>无</td><td></td></tr><tr><td> NAD </td><td> 175毫米，pH值7.5-8 </td><td> 300微升</td><td>无</td><td>为了达到PH值7.5-8，滴定用Tris在2 M。 </td></tr><tr><td>腺苷</td><td> 650毫米，pH值为8 </td><td> 200微升</td><td>无</td><td>为了达到pH值为8，用滴定三在2 M。 </td></tr><tr><td>亚叶酸</td><td> 33.9毫</td><td> 300微升</td><td>无</td><td>虽然只有300微升是必要的，配方中补充是1.15毫升。 </td></tr><tr><td>亚精胺</td><td> 1个月</td><td> 150微升</td><td>无</td><td>储存在4℃下，加热至37°C至融化。 </td></tr><tr><td> 3-PGA </td><td> 1.4 M，pH值7.5 </td><td> 3.2毫升</td><td>无</td><td>以达到pH为7.5，滴定用Tris于2 M。 </td></tr></tbody></table> 表4。试剂的能源解决方案的协议做准备。 参考文献1。食谱的项目。 氯霉素，34毫克/毫升：准备0.51克氯霉素加乙醇至15ml。过滤消毒（0.22微米）等分至1ml管，于-20℃供以后使用商店。 的2xYT + P +厘米琼脂板：准备1.24克的2xYT，1.6毫升磷酸氢二钾溶液@ 1 M，0.88毫升磷酸二氢钾溶液@ 1米，0.6克琼脂和水至40ml。高压灭菌。放冷至50℃，加入40微升厘米。分装25毫升成100×15毫米培养皿中，放凉了一个小时。 的2xYT + P介质：准备124克的2xYT，160毫升磷酸氢二钾溶液@ 1 M，88毫升磷酸二氢钾溶液@ 1 M和水为4L Aliqu加时赛出到2×1.88 L和0.24 L。高压灭菌。 Tris碱，2M：准备60.57克Tris碱和水至250 mL。消毒，储存在室温下以备后用。 DTT，M：准备2.31 ​​克DTT和水至15ml。过滤消毒（0.22微米），分装至1 ml离心管，于-20℃以备后用商店。 S30A缓冲区：准备10.88克的Mg-谷氨酸和24.39克谷氨酸钾50毫升的Tris在2M，乙酸（pH值7.7），和水2升高压釜中，在4℃下储存，加入4 mL 1 M DTT使用前。 S30B缓冲区：准备10.88克的Mg-谷氨酸和24.39克谷氨酸钾，三为2米（pH值8.2），和水2升高压釜中，在4℃下储存，加2毫升的1摩尔使用前DTT。 HEPES：准备1.91克HEPES（分子量238.21），KOH（pH为8），和水到4ml。 氨酰tRNA：准备30毫克的tRNA和水至600μL。 辅酶A：准备30毫克辅酶A（分子量767.53）和水至600μL。 NAD加入 34.83毫克的NAD（MW 663.43），三在2 M（至pH 7.5-8）和水到300微升。 （加入27微升的Tris中的2 M来使溶液至pH 7.5-8）。 营：新增42.80毫克的cAMP（MW 329.22），三为2米（至pH为8），和水至200μL。 （加入73微升的Tris中的2 M来使溶液至pH为8）。 亚叶酸（33.9毫米）：为20毫克固体亚叶酸钙盐（分子量511.5），加1.15毫升水。 亚精胺：准备23.55微升亚精胺（分子量145.25）和水至150μL。在室温在37°C短暂熔化后准备 3-PGA：添加1.03克3-PGA（分子量230.02）的液，Tris在2米（pH为7.5）和水至320毫升（添加1.73的Tris中的溶液在2 M来使溶液pH为7.5）。 NucleotIDE混合：加入145毫克的三磷酸腺苷二钾盐二水（619.4兆瓦），133毫克的GTP二钠盐（MW 567.14），79.4毫克CTP二钠盐（分子量563.16），82.6毫克的类UTP三钠盐二水（分子量586.12） ，氢氧化钾的15％稀释液（pH为7.5），以及水至1.5毫升（加入353微升的KOH在15％稀释，使溶液pH为7.5）。 补充材料2。 Bradford法。 <ol><li>从4℃取出布拉德福德剂，并设置在室温下。 </li><li>制备50微升BSA标准在1毫克/毫升和0.1毫克/毫升。 </li><li>准备40微升20倍稀释提取物从步骤1.47。 </li><li>加入800μl水至7比色皿。 </li><li>制备标准比色皿为0毫克/毫升，1毫克/毫升（10微升0.1毫克/毫升BSA），2毫克/毫升（20微升0.1毫克/毫升BSA），4毫克/毫升（4微升1毫克/毫升BSA） ，6毫克/毫升（6微升1毫克/毫升BSA）。 </li><li>准备实验皿2微升的样品和4微升的样品。 </li><li>加入200μl布拉德福德剂到每个试管混匀B井Ÿ移液。在室温下孵育至少10分钟。 </li><li>制作标准曲线使用比色皿从步骤6.5外径595纳米。拒绝标准曲线如果r 2 &lt;0.95。 </li><li>确定提取物浓度使用比色皿从步骤6.6外径595纳米。 </li></ol>补充材料3。缓冲校准电子表格。 <a href="https://www.jove.com/files/ftp_upload/50762/TXTL_e(template)_calibration_JoVE.xlsx" target="_blank">见TXTL_e（模板）_calibration_JoVE.xlsx</a> 。 补充材料4。无细胞表达运行电子表格。 <a href="https://www.jove.com/files/ftp_upload/50762/TXTL_JoVE.xlsx" target="_blank">见TXTL _JoVE.xlsx</a> 。

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

这次为期五天的协议列出了所有步骤，设备，以及用于创建和运行基于一个有效的内源性大肠杆菌从头TX-TL无细胞表达系统必要的补充软件。用试剂，该协议需要8个小时或更短设置一个反应，收集和处理数据。

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 ...

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

62.7K 次观看.  California Institute of Technology. 这次为期五天的协议列出了所有步骤，设备，以及用于创建和运行基于一个有效的内源性大肠杆菌从头TX-TL无细胞表达系统必要的补充软件。用试剂，该协议需要8个小时或更短设置一个反应，收集和处理数据。 

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

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

科研

生物学

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

对于电感受的制备快速协议<em&gt;大肠杆菌</em&gt;和<em&gt;霍乱弧菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.