E.G. Worst and A. Ott acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) within the collaborative research center SFB 1027 as well as Saarland University. E.G. Worst, A. Ott and V. Noireaux further acknowledge financial aid by the Human Frontiers Science Program Organization (HFSPO). The authors thank Tobias Baumann and Stefan Oehm (Institute of Chemistry, Technische Universit&#228;t Berlin) for critical reading.

The authors have nothing to disclose.

安易于使用的无细胞表达系统作为一个可行的策略，去渣特异性结合NCAA锦标赛成蛋白质，提出。为此，将粗提取物补充有载体DNA编码所关注的相应氨基酸的蛋白质，能量缓冲器和。需要注意的是粗提取物等分试样体积取决于粗提取物的蛋白质浓度34。无细胞表达效率取决于载体DNA构建浓度优化。能量缓冲器组件的体积为优化MG-和K-谷氨酸浓度的函数，以使所述无细胞表达模型蛋白的高产率而有所不同。 掺入实验的初步评估可以通过执行未纯化无细胞反应介质的SDS-PAGE来获得。有关更详尽的分析，HPLC-ESI质谱分析，提出为手段，以检查是否有完整的，具体的残留INCOR在NCAA的穿孔。至于后者的制备，旋转柱系统用于使His标签纯化和缓冲与我们在这个协议中使用小体积交换。 包括HPLC-ESI质谱，整个协议可在2天内进行。它不包括任何特别关键的步骤。然而，MG-和K-谷氨酸浓度的优化以及载体的DNA是为了表达的模型蛋白的高产率是至关重要的。使用高效表达载体的个体极值-OR 2-OR 1-PR-UTR1-gene_of_model_protein-T500强烈推荐。 His-标记的蛋白的洗脱通常是由于高浓度的咪唑（&gt; 150毫米）和其他盐如NaH 2 PO 4（&gt; 300毫米）或氯化钠（&gt; 50毫米）产生在质谱分析49高背景噪声。用合适的蛋白存储缓冲器这样的洗脱缓冲液的交换稳定模型prote在和大幅降低质谱分析中的背景噪声。 其结果是，可在模型蛋白质内的所有六个位置替换精氨酸。在表达系统中，没有精氨酸残基可以被检测出来。这简化相比，需要进一步消耗策略29,30其他表达系统精氨酸类似物的特定残基掺入。所提出的无细胞的方法规避那些因灿毒性在体内的方法存在固有局限性，或在单一蛋白生产策略24,31 mRNA序列的强烈依赖。相反， 在体外系统中采用，体内灿裂解丝氨酸和羟基胍确实发生31。 但是，无细胞系统保留足够量的Lys与类似物如HYL竞争。液相色谱 - ESI质谱分析表明，该模型蛋白含有两者，的Canonical以及不同比例的非规范类似物。赖氨酸的特定残基掺入是在一般可能的，但对于完全取代，进一步消耗策略，或特别设计的aaRS和tRNA的识别NCAA锦标赛的优化需要开发。 我们在相同的浓度为规范那些加入NCAA获得的无细胞表达，修饰的模型蛋白的优良的产率。掺入效率取决于NCAA的性质被并入。甚至更高的收益率可能仍然是通过优化NCAA的浓度实现的。 只要它们是由典型的内源性翻译系统接受所呈现的结果证明了NCAA锦标赛的特定残基掺入所采用的系统的适用性。对于特定NCAA锦标赛的特定残基掺入，一个另外的需求，以检查是否类似CAA distur的残B中的表达系统。 无细胞转录-翻译系统可以从不同的生物体进行工程化，以不同的需求54作出响应。所有E.在这里提出的无细胞系统的大肠杆菌转录-翻译机器使噬菌体和大肠杆菌的使用大肠杆菌的启动子，它们可以并行地或连续地在级联55采取行动。的普遍适用性和可用性使该方法在氨基酸毒性和治疗应用的进一步研究的有力工具。

遗传密码是通用的生物圈。它的代码的一组20种经典氨基酸，有时由硒代1或吡咯2延长。这是转换的帮助的tRNA的成折叠成蛋白质的氨基酸链的遗传密码的核糖体。规范氨基酸的官能团，与翻译后修饰组合，向一个非常广泛的蛋白质功能3,4。原则上，由于该组有限的经典氨基酸功能限制可以通过进一步结合来克服，非规范氨基酸（NCAA锦标赛），使新的化学和新的功能3,4-。 还有的NCAA锦标赛的结合两种互补的方法:在定点或特定渣掺入。前一种方法需要大量的技术难题，因为规范的一套氨酰-tRNA合成器的etases（的aaRS）和tRNA的必须由正交的aaRS酰tRNA对，必须不与内源性翻译机制相互作用进行扩展。基于仔细的工程，这种方法采用了NCAA锦标赛截至所需的蛋白质站点的单点突变。 NCAA锦标赛的位点特异性掺入基因由未分配给典型氨基酸（CAA），通常是一个终止密码子5-9的密码子编码。这种方法需要在给定的网站在功能上的变化，而 ​​不是整个蛋白10-13。 相反，具体的残基掺入依赖于规范翻译机器错误识别的非规范氨基酸。掺入的发生是由于缺乏的aaRS的底物特异性。 NCAA锦标赛的特定残基掺入，建立在科恩和同事14的工作，导致了重要的应用3,10，其中蛋白质的生物正交标记15-17或X射线晶体18蛋白质的结构解析。 由于天然的aaRS一般都喜欢在一个同构NCAA其同源的氨基酸，体内特定渣掺入效率通常需要营养缺陷型表达宿主不能合成NCAA的规范模拟。的宿主细胞培养在生长培养基中，可提供仅类似CAA的低浓度。它与补充的连续与NCAA组合枯竭迫使表达宿主纳入了NCAA到模型中的蛋白质在多个，规范地规定站点。与此相反的位点特异性的方式，这通常对整个蛋白结构的深的影响，从而导致相当修饰的蛋白质19,20的物理和化学性质。然而，大多数的NCAA锦标赛的是表达宿主3生长抑制剂，因为它们被结合到许多其他PROT除了重组基因表达过程中的利益EINS。这清楚地限制了体内的方法。的氨基酸，是有毒或对蛋白质结构强的影响的体内掺入仍然特别具有挑战性。然而，这些分子的工程师具有非凡的功能的蛋白质中最有前途的。 一个例子是有毒的，非规范，天然存在的L-刀豆氨酸（CAN），L-精氨酸（Arg）的类似物。它影响和块ARG有关的调节和催化反应途径，以及其在活细胞的存在可以导致立即死亡3,21-23。其纳入蛋白质精氨酸在位置可以减少蛋白质的稳定性21-23。由于所得的毒性，含有在大肠杆菌 （ 大肠杆菌 ）和其它普通的表达宿主的蛋白质刀豆氨酸的表达仍然是一个挑战。由于这些原因，完整的体内我灿ncorporation在所有的精氨酸位置已经恰当地被证实只有一次24，使用单阐述蛋白质的生产体系。然而，也已经被提出作为一种抗癌剂25-27，并作为用于人28自身免疫性疾病的刺激。此外，它受到在其抗代谢，抗细菌，抗真菌的各种研究和抗病毒特性25的。这些特性提高了需求高效，​​易于执行的方法来表达可含有蛋白质为药用，医学和功能研究。 虽然连接到体内生产的许多问题可以使用无细胞表达系统来回避， 在体外特异性残基的办法只被很差探讨。已报道的L-色氨酸类似物29和多个NCAA锦标赛30的无细胞特异性残基结合。这些方法是基于高度efficient T7 RNA聚合酶。 T7 RNA聚合酶嗣继承噬菌体样转录，从而减少在比较源性转录遗传功能。 罐的完整特定残基掺入模型蛋白在所有精氨酸位置最近报道31，使用无细胞表达系统32。同一系统的一个稍微的改变通过停止启用不同的吡咯类似物的位点特异性并入模型蛋白抑制密码子33。所采用的无细胞系统31 - 33是基于一个所有大肠杆菌大肠杆菌转录-翻译系统。然而，它使蛋白表达尽可能有效电流噬菌体系统（0.5 -为1mg / ml重组蛋白）32，同时保留大部分的原始转录-翻译模块化。 在这项工作中，一个详细的协议被提供关于如何渣油NCAA锦标赛的UE特定掺入可实现，使用此所有大肠杆菌大肠杆菌无细胞系统32。此外，提出进一步的措施，准备通过HPLC-ESI质谱恰当的评价表达的蛋白质。为了扩大这种无细胞系统的特性，这项工作并不仅仅指灿31日公布的合并，但也提出相关的非经典的L-赖氨酸模拟L-羟基赖氨酸新的数据。 为NCAA锦标赛的特定残基掺入下列协议是最近发表在朱庇特34的协议的适应。后者协议描述如何执行与标准氨基酸高效无细胞表达。此外，它提出了粗无细胞提取物的制备中，氨基酸溶液，能量储备溶液，并在该方法中使用的能量缓冲器。以下方案相比集中在改性步骤将前面Protocol为了使NCAA锦标赛的特定残基掺入。校准的移液管，低结合枪头和微离心管建议的准备。在下面，使用IUPAC缩写为氨基酸。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protective eyewear</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z758841</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves (size S)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z768960</td>
			<td>Catalog numbers other sizes: Z768979 for M, Z768987 for L and Z768995 for XL</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tube 1.5 ml (PCR clean)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>30123.328</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbalance Discovery DV114CM</td>
			<td>Ohaus, Greifensee, Switzerland</td>
			<td>80104140</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspatula (L 6 5/8 in., stainless steel, rod diam. 0.09 in.)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z243213</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Canavanine</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>C9758</td>
			<td>Acute toxicity: wear eyeshields, dust mask, protective gloves</td>
		</tr>
		<tr>
			<td>Hydroxylysine (racemic mixture)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>H0377</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo-gloves (size S, water resistent)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z183490</td>
			<td>Catalog numbers other sizes: Z183512 for M, Z183520 for L and Z183539 for XL</td>
		</tr>
		<tr>
			<td>RTS Amino Acid Sampler</td>
			<td>Biotechrabbit, Hennigsdorf, Germany</td>
			<td>BR1401801</td>
			<td>For homemade preparation of amino acid stock solutions, follow this protocol35 and use the solid amino acid kit LAA21-1KT, L-proline (81709-25G), L-cysteine (30089-25G), L-histidine (53319-25G) and L-lysine (L5501-5G)&#160; (all from Sigma-Aldrich, St. Louis, USA)</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>H6147</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>A8937</td>
			<td></td>
		</tr>
		<tr>
			<td>CTP</td>
			<td>Affymetrix, Santa Clara, USA</td>
			<td>14121</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Affymetrix, Santa Clara, USA</td>
			<td>16800</td>
			<td></td>
		</tr>
		<tr>
			<td>UTP</td>
			<td>Affymetrix, Santa Clara, USA</td>
			<td>23160</td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA (from E. coli, pack size 100 mg)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>10109541001</td>
			<td>Catalog number for pack size of 500 mg is 10109550001</td>
		</tr>
		<tr>
			<td>CoA</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>C4282</td>
			<td></td>
		</tr>
		<tr>
			<td>NAD (from yeast )</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>N6522</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>A9501&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>F7878</td>
			<td></td>
		</tr>
		<tr>
			<td>3-PGA</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>P8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg-glutamate</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>49605</td>
			<td></td>
		</tr>
		<tr>
			<td>K-glutamate</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>G1149&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>pBEST-OR2-OR1-Pr-UTR1-deGFP-T500</td>
			<td>Addgene, Cambridge, USA</td>
			<td>Plasmid #40019</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20 % precast Tris-Glycine Gels (10 cm x 10 cm x 1 mm, 10 courses)</td>
			<td>Anamed Elektrophorese, Gro&#223;-Bieberau / Rodau, Germany</td>
			<td>TG 81610</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS running buffer (10 x concentrate, 5000 ml)</td>
			<td>Anamed Elektrophorese, Gro&#223;-Bieberau / Rodau, Germany</td>
			<td>TG 50001</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS loading buffer (2 x concentrate, 50 ml)</td>
			<td>Anamed Elektrophorese, Gro&#223;-Bieberau / Rodau, Germany</td>
			<td>TG 05002</td>
			<td></td>
		</tr>
		<tr>
			<td>Unstained protein marker, broad range (2-212 kDa)</td>
			<td>New England Biolabs, Ipswich, USA</td>
			<td>P7702S</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1060091011</td>
			<td>Toxic by inhalation, in contact with skin and if swallowed: wear protective gloves and work under fume hood</td>
		</tr>
		<tr>
			<td>Acetic acid (99.8 %)</td>
			<td>VWR International, Darmstadt, Germany</td>
			<td>20104.447</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Blue G-250 (10 g)</td>
			<td>Biozym Scientific, Hessisch Oldendorf, Germany</td>
			<td>902120</td>
			<td></td>
		</tr>
		<tr>
			<td>His-Spin Protein Miniprep kit</td>
			<td>Zymo Research Europe, Freiburg, Germany</td>
			<td>P2002</td>
			<td>Product also distributed by Zymo Research Corporation, Irvine, USA&#160;</td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol, 99 %</td>
			<td>VWR International, Darmstadt, Germany</td>
			<td>24397.296DB</td>
			<td></td>
		</tr>
		<tr>
			<td>CentriPure Z25 mini spin columns</td>
			<td>Genaxxon bioscience, Ulm, Germany</td>
			<td>CP-0205-Z100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrator 5301</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>5301 000.210</td>
			<td></td>
		</tr>
		<tr>
			<td>2xYT</td>
			<td>MP biomedicals, Santa Ana, USA</td>
			<td>113012032</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Agar</td>
			<td>BD Diagnostics, Franklin Lakes, USA</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead-beating tubes (polypropylene microvials)</td>
			<td>Biospec, Bartlesville, USA</td>
			<td>522S</td>
			<td></td>
		</tr>
		<tr>
			<td>Beads, 0.1mm dia.</td>
			<td>Biospec, Bartlesville, USA</td>
			<td>11079101</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 Rosetta 2 E. coli strain</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>71402</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford BSA protein assay Kit</td>
			<td>Bio-Rad, M&#252;nchen, Germany</td>
			<td>500-0201</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>C1919</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes, 1.5ml</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>14-955-127</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Bio-Spin Chromatography Columns</td>
			<td>Bio-Rad, M&#252;nchen, Germany</td>
			<td>732-6204</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 384-well optical bottom plates</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>142761</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc sealing tape</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>232701</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>Promega, Madison, USA</td>
			<td>V3011</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic solution</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>P8584</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic solution</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>P8709</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer dialysis cassettes, 10k MWCO (Kit)</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>66382</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>85558</td>
			<td></td>
		</tr>
		<tr>
			<td>1L centrifuge bottle</td>
			<td>Beckman-Coulter, Brea, USA</td>
			<td>A98813</td>
			<td></td>
		</tr>
		<tr>
			<td>4L Erlenmeyer flask</td>
			<td>Kimble Chase, Vineland (NJ), USA</td>
			<td>26500-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-26XP centrifuge</td>
			<td>Beckman-Coulter, Brea, USA</td>
			<td>393127</td>
			<td>Or centrifuge equivalent being able to centrifuge 1 l bottles.</td>
		</tr>
		<tr>
			<td>Forma 480 orbital shaker</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>480</td>
			<td>Or shaker equivalent being able to shake chest-size 6 x 4 L .</td>
		</tr>
		<tr>
			<td>JLA-8.1000 rotor</td>
			<td>Beckman-Coulter, Brea, USA</td>
			<td>363688</td>
			<td>Or 5000 x g rotor equivalent for above centrifuge equivalent being able to centrifuge 1L-bottles.</td>
		</tr>
		<tr>
			<td>Mini-Beadbeater-1</td>
			<td>Biospec, Bartlesville, USA</td>
			<td>3110BX</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge 22R refrigerated microcentrifuge</td>
			<td>Beckman-Coulter, Brea, USA</td>
			<td>368831</td>
			<td>Or centrifuge equivalent being able to centrifuge 2 ml reaction tubes.</td>
		</tr>
		<tr>
			<td>Heating block HLC HBT 130</td>
			<td>Labexchange, Burladingen, Germany</td>
			<td>24465</td>
			<td>Or heating block equivalent being able to heat samples in reaction tubes up to 100 &#176;C</td>
		</tr>
		<tr>
			<td>Eppendorf MiniSpin centrifuge</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>5452000018</td>
			<td>Or centrifuge equivalent being able to centrifuge 2 ml reaction tubes.</td>
		</tr>
		<tr>
			<td>IKA Vortex 3 (4 mm orbital shaker diameter, 0 - 2500 rpm)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z654760</td>
			<td>Or vortex equivalent</td>
		</tr>
		<tr>
			<td>Scotsman AF103 ice flaker machine</td>
			<td>K&#228;lte-Berlin, Berlin, Germany</td>
			<td>AF103</td>
			<td>Or ice flaker machine equivalent</td>
		</tr>
		<tr>
			<td>MyTemp mini digital incubator</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z763314</td>
			<td>Or incubator equivalent being able to heat samples at 29 &#176;C</td>
		</tr>
		<tr>
			<td>EcoCell electrophoresis cell / chamber</td>
			<td>Anamed Elektrophorese, Gro&#223;-Bieberau / Rodau, Germany</td>
			<td>AN12005</td>
			<td>Or electrophoresis chamber equivalent being able to perform vertical gel electrophoresis with above precast gels or other used gels</td>
		</tr>
		<tr>
			<td>Power-phor power supply for electrophoresis cell / chamber</td>
			<td>Anamed Elektrophorese, Gro&#223;-Bieberau / Rodau, Germany</td>
			<td>AN12001</td>
			<td>Or power supply equivalent being able to supply above used electrophoresis cell / chamber with power</td>
		</tr>
		<tr>
			<td>VWR Signature Ergonomic High Performance Single-Channel Variable Volume Pipettors Starter Kit 1</td>
			<td>VWR International, Darmstadt, Germany</td>
			<td>613-5278</td>
			<td>Or equivalent micropipettes enabling to pipette similar volumes with the same precision</td>
		</tr>
		<tr>
			<td>VWR Signature Ergonomic High Performance Single-Channel Variable Volume Pipettors Starter Kit 2</td>
			<td>VWR International, Darmstadt, Germany</td>
			<td>613-5279</td>
			<td>Or equivalent micropipettes enabling to pipette similar volumes with the same precision</td>
		</tr>
		<tr>
			<td>Pipetman Tips Diamond D10ST (0.1 - 10 &#181;l)</td>
			<td>Gilson, Middleton, USA</td>
			<td>F171101</td>
			<td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td>
		</tr>
		<tr>
			<td>Pipetman Tips Diamond D200ST (2 - 200 &#181;l)</td>
			<td>Gilson, Middleton, USA</td>
			<td>F171301</td>
			<td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td>
		</tr>
		<tr>
			<td>Pipetman Tips Diamond D1000ST (100 - 1000 &#181;l)</td>
			<td>Gilson, Middleton, USA</td>
			<td>F171501</td>
			<td>Or equivalent low-binding pipette tips enabling to pipette similar volumes with the same precision</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tubes with screw caps (sterile)</td>
			<td>VWR International, Darmstadt, Germany</td>
			<td>50-0156</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tubes with screw caps (sterile)</td>
			<td>VWR International, Darmstadt, Germany</td>
			<td>525-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml polypropylene tubes (round bottom, two-position vent stopper, sterile)</td>
			<td>Greiner Bio-One, Frickenhausen, Germany</td>
			<td>187262</td>
			<td></td>
		</tr>
		<tr>
			<td>Discovery BIO Wide Pore C5 HPLC Column (3 &#181;m particle size, L x I.D. 10 cm x 2.1 mm)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>567227-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1260 HPLC machine&#160;</td>
			<td>Agilent Technologies, Santa Clara, USA</td>
			<td>G1312B</td>
			<td></td>
		</tr>
		<tr>
			<td>6500 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS</td>
			<td>Agilent Technologies, Santa Clara, USA</td>
			<td>G6530BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile&#160;</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>270717</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOstar Omega microplate reader</td>
			<td>BMG Labtech, Ortenberg, Germany</td>
			<td>415-101</td>
			<td>Or microplate reader equivalent being able to measure the fluorescence of the expressed model protein</td>
		</tr>
		<tr>
			<td>Hanna Checker pH meter</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>Z351091&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid eluent additive for LC-MS</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>56302</td>
			<td></td>
		</tr>
	</tbody>
</table>

residue-specific incorporation of noncanonical amino acids into model proteins using an escherichia coli cell-free transcription-translation system

的标准集氨基酸导致了一个非常广泛的蛋白质的功能。然而，该组的残基仍对潜在的蛋白的应用的限制。非规范的氨基酸的结合可以扩大这个范围。有用于非规范氨基酸结合两种互补的方法。为位点特异性掺入，除了内源性的规范平移机械，正交氨酰基-tRNA合成酶氨酰tRNA对必须提供不与规范那些交互。因此，未分配到典型的氨基酸，通常是一个终止密码子的密码子，也是必需的。此遗传密码膨胀使非经典氨基酸的掺入在蛋白质内一个单一的，给定的位点。在这里提出的工作描述了特定残基掺入，其中遗传密码是内源性的翻译系统内重新分配。翻译机器一ccepts的非规范氨基酸作为替代在标准地规定的地点， 即掺入它，在该蛋白质的典型氨基酸出现的所有被非规范取代。的非规范氨基酸掺入可以改变蛋白质的结构，从而引起相当改性的物理和化学性质。非规范氨基酸类似物常常充当细胞生长抑制剂表达宿主，因为它们修改内源性蛋白， 在体内蛋白质生产限制性的。 在体内的毒性非规范氨基酸掺入蛋白质仍然特别具有挑战性。这里，对于由非经典氨基酸L-刀豆氨酸的完全替代L-精氨酸的无细胞的方法，提出。它绕过体内表达的固有的困难。此外，包括准备靶蛋白进行质谱分析的协议。它表明，L-赖氨酸可以通过将L-羟基赖氨酸取代，尽管具有较低的效率。原则上，任何非规范氨基酸类似物可以使用所提出的方法，只要内源性体外翻译系统识别它被并入。

此协议通过NCAA锦标赛的无细胞特异性残基掺入模型蛋白质指导。它提出了纳入实验，并进一步步骤准备模型蛋白质的适当HPLC-ESI质谱分析的初步评估SDS-PAGE。 这里，精氨酸类似物可以的无细胞特异性残基掺入，以及赖氨酸模拟L - 羟基赖氨酸（HYL）的代表性结果。不同的氨基酸溶液，能量缓冲器，如上所述制备的模型蛋白和无细胞反应的载体DNA编码。参考无细胞反应被提供有由所述20中国农业科学院的氨基酸溶液。对于每一个实验中，阴性对照无细胞反应用缺乏在NCAA的问题的典型的模拟的氨基酸溶液供给。对于每个APPRoach一无细胞反应表达在氨基酸溶液，其中该CAA是由非规范模拟取代的存在下模型蛋白。 His标签纯化，缓冲液交换和HPLC-ESI质谱分析是按照上述的协议执行。 该模型蛋白是C-末端His标签的deGFP 32，EGFP 53的截短形式。其单字母氨基酸序列可以在（补充材料2）中找到。该模型蛋白含有6 Arg和18赖氨酸的位置， 分别表达载体是的pbest-OR 2-OR 1-PR-UTR1-deGFP-T500。 在NCAA的完全结合的情况下，可以假设的阴性对照反应不表达deGFP，由于20 CAAS缺失中的一个。相反，deGFP必须在其它两个反应检测:在refe天然1伦斯无细胞反应，并在被提供与NCAA无细胞反应修饰蛋白。 图1A示出了制罐掺入实验的初步的SDS-PAGE评估。参考无细胞反应具有最高deGFP表达水平。在被设置有可在无细胞反应，deGFP在稍低浓度表示。没有deGFP表达所用的阴性对照来检测。此的SDS-PAGE的结果是对罐的一个成功的掺入靶蛋白deGFP的良好指示。 为了证明制罐的假设完整纳入deGFP，既纯化模型蛋白质，在图1B可见，通过HPLC-ESI质谱进行分析。 图1C示出了纯化的deGFP分子的解卷积质谱。 deGF的解卷积质量<html>即在参考无细胞反应表示P是26,192.8道尔顿。对于deGFP在含有无细胞反应出现26202.5大的质量的制罐表达。对于本地deGFP6Arg和精氨酸修饰deGFP6Can预期由群众在被全面取代是26193 Da和26204大分别。 1.5大的为deGFP6Can的质量差是光谱去卷积的误差范围内。因此，能充分纳入deGFP在所有6精氨酸位置被确认。 减少强度的两个峰值对应于天然deGFP6Arg和改性deGFP6Can未达到其成熟荧光团。荧光团是自催化通过消除一个水分子的产生，接着氧化。这导致了质量提高了20大，如果这个过程无法继续。 PG"/&gt; 图 1. 无细胞表达和纯化deGFP分子可以掺入实验和HPLC-ESI质谱的SDS-PAGE评估。（A）中使用SDS-PAGE的能掺入实验的初步评价。从左至右依次为:蛋白质标准，标准无细胞反应，阴性对照和无细胞反应提供可替代精氨酸。 His标签纯化后（B）的SDS-PAGE和缓冲表示deGFP分子交换。从左至右依次为:蛋白质标准，从参考反应提纯deGFP，由含灿反应纯化deGFP。 （C）采用HPLC-ESI质谱灿全部并入的确认。本地deGFP和精氨酸修饰deGFP的预期由群众完全可以取代是26193 Da和26204大分别。每个光谱被归一化到其最高强度（计数）。峰值位置指示在大。为可视化的目的，将凝胶泳道被从凝胶图片提取的，连接在一起，被转换成灰度格式，大小被优化和对比度以及亮度增强。原厂胶道在补充材料3介绍<a href="https://www.jove.com/files/ftp_upload/54273/54273fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2A示出了HYL掺入实验的初步的SDS-PAGE评估。在于设置有HYL无细胞反应，deGFP表示。相反于第一实验中，将弱deGFP带所用的阴性对照反应中观察到。这可能是由于在无细胞反应赖氨酸残基。这使得在阴性对照反应，其中加入既不赖氨酸也不HYL微弱deGFP表达。 用于h<html> PLC-ESI质谱，设置有HYL无细胞反应的deGFP分子是纯化的和交换缓冲液（ 图2B）。 <img alt="图2" src="/files/ftp_upload/54273/54273fig2.jpg" /> 图2. SDS-PAGE中掺入HYL实验的评价 （A）从左至右依次为:阴性对照无细胞反应，含HYL和蛋白质的标准无细胞的反应。 His标签纯化后（B）的SDS-PAGE和缓冲表示deGFP分子交换。从左至右依次为:含反应和蛋白质纯化标准从deGFP的HYL。为可视化的目的，将凝胶泳道被从凝胶图片提取的，连接在一起，被转换成灰度格式，大小被优化和对比度以及亮度增强。原厂胶道在补充材料3中。F ="htt​​ps://www.jove.com/files/ftp_upload/54273/54273fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 图3示出纯化deGFP分子的解卷积质谱。 图3A证实在无细胞的反应已经存在赖氨酸残基的假说。频谱的主要峰对应于天然deGFP（预期质量:26193道尔顿）。再次，一个20达较高的质量未开发其荧光团deGFP分子可以被检测出来。的赖氨酸残基被优先加载到tRNA的赖氨酸由赖氨酰tRNA合成酶，导致天然deGFP18Lys的高表达水平。 HYL和Lys之间的质量差是16达。由于HYL的存在是在竞争的赖氨酸残基被生成的所有可能的deGFP物种（deGFP18Hyl，deGFP17Hly + 1Lys，...，deGFP16Hyl + 2Lys）（ 图3B）。诚然，deGFP1Hyl + 17Lys的峰值与没有产生其荧光（ 图3A）和一些峰的质谱与预期质量相差超过2达天然deGFP的峰值重叠。这些质量的差异可以归因于高噪音由于低量的deGFP物种。然而，HYL一般是由无细胞系统并入。进一步改进必须完成取消在无细胞反应赖氨酸残基。 <img alt="图3" src="/files/ftp_upload/54273/54273fig3.jpg" /> 图 3. HPLC-ESI质谱含有HYL无细胞反应的纯化deGFP分子光谱 （A）中本机deGFP18Lys主要是检测到（预期群众:用荧光团26193道尔顿，而不成熟荧光团26213道尔顿）。 （B）放大倍数显示所有可能deGFP种（deGFP18Hyl，deGFP17Hly + 1Lys，deGFP16Hyl + 2Lys，...，deGFP1Hyl + 17Lys）的存在。其预期群众26193大+ N×16大（N = 1，...，18）。频谱归一化到其最高强度（计数）。峰的位置以达表示。 <a href="https://www.jove.com/files/ftp_upload/54273/54273fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/54273/54273supfig1.jpg" /> 补充材料1.计算模板。在部分2中，能量缓冲器主混合物的制备是用最佳MG-30微升和粗提物的等分试样，并分别为3毫米的K-谷氨酸浓度和30mM的举例说明。这个例子导致了产生3个能量缓冲等分预混量。在秒化3，无细胞反应的制备采用90纳米DNA原液导致无细胞反应的10纳米的最佳载体DNA浓度例证。 <a href="https://www.jove.com/files/ftp_upload/54273/cell-free_calculations_JoVE_11.03.2016_final.xlsx" target="_blank">请点击此处下载此文件。</a> 粗提取物体积:28微升，最佳的Mg谷氨酸:2毫米，最佳的K-谷氨酸:40毫米，编号为适当的可追溯性，使用计算模板是由从上面的例子不同，这些典型值的插入例举的所需的缓冲区等份:100，在无细胞反应最佳矢量浓度:8纳米，DNA载体原液:150纳米。 首先进入28微升的粗提取物量进入第一次模板部分的橙色领域。然后，进入到第二模板部2mm的D 40毫米为最佳MG-和K-谷氨酸浓度为橙色领域。考虑到最佳MG-和K-浓度，15微升能量缓冲液的组成，以及一个相应的，按比例增加16微升等分试样被计算。下面，相应输入100作为能量缓冲器等分的期望数目（16微升）。模板适应为1,700微升主结构不同的缓冲部件的体积为:204微升100毫米镁谷氨酸原液，136微升3M的K-谷氨酸原液，728.73微升14倍的能源解决方案，510微升的40％PEG-8000和121.27微升无菌DDH 2 O最后，在第三模板部分，输入8 50nm和150nm作为无细胞反应最佳矢量浓度分别载体DNA原液浓度。模板适应必须被添加到28微升粗提取物的不同组分的量以完成一个90微升的制备无细胞反应如下:15微升能量缓冲液，15微升3不同组成的氨基酸溶液之一，4.80微升的载体DNA溶液（150纳米），和27.20微升无菌DDH 2 O的<img alt="图2" src="/files/ftp_upload/54273/54273supfig2.jpg" /> 补充材料2.一个字母的模型蛋白deGFP的氨基酸序列。这个模型蛋白含有6精氨酸和赖氨酸18立场。 <a href="https://www.jove.com/files/ftp_upload/54273/54273supfig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54273/54273supfig3.jpg" /> 补充材料3，全长度和对应于该凝胶图片未修改凝胶泳道呈现图1和2中的凝胶泳道中的每个的各个S呈现<html> ubfigure由相同的SDS聚丙烯酰胺凝胶中提取。在图1和2 中这些泳道进行演示接合在一起。蛋白质标准带的分子量的数字旁边指出。 （A.1） 图1A的未裁剪胶道。从左至右依次为:蛋白质标准，标准无细胞反应，阴性对照和无细胞反应提供可替代精氨酸。 （A.2） 图1B的未裁剪胶道。从左至右依次为:蛋白质标准，从参考反应提纯deGFP，由含灿反应纯化deGFP。 （B.1） 图2A的未裁剪胶道。从左至右依次为:阴性对照无细胞反应，含HYL和蛋白质的标准无细胞的反应。 （B.2） 图2B的未裁剪胶道。从左至右依次为:含反应和蛋白质纯化标准从deGFP的HYL。D / 54273 / 54273supfig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System at JoVE.co

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.

非经典氨基酸的具体残留纳入到模型蛋白质使用<em&gt;大肠杆菌</em&gt;无细胞转录翻译系统

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

residue-specific-incorporation-noncanonical-amino-acids-into-model

Research

JoVE Journal

Biology

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

15.9K 次观看.  Saarland University. 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.

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

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

科研

生物学

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

一步代谢：碳水化合物，在一个单一的过程量化的有机和氨基酸

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

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 ...

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 ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

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 ...

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 ...

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. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...