Name: preparing protein producing synthetic cells using cell free bacterial extracts, liposomes and emulsion transfer
Uploaded: 2020-04-27T13:00:00
Description: Watch this Scientific Journal Video about Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer at JoVE.com

这项工作得到了ERC-STG-2015-680242的支持。
作者还承认技术综合癌症中心（TICC）的支持;罗素浆果纳米技术研究所;罗瑞一·洛基生命科学与工程跨学科中心;以色列经济部为卡明赠款（52752）;以色列科学技术和空间部首席科学家办公室（3-11878）;以色列科学基金会（1778/13，1421/17）;以色列癌症协会（2015-0116）;德国-以色列科学研究与发展基金会为GIF青年赠款（I-2328-1139.10/2012）;欧洲联盟FP-7职业一体化赠款IRG方案（908049）;磷脂研究中心赠款;罗森布拉特癌症研究基金会，马拉特家庭基金会赠款;和昂格家庭基金会施罗德承认阿隆和陶布奖学金。O. Adir承认谢尔曼和古特维尔斯奖学金。陈承认谢尔曼奖学金。克林斯基感谢罗斯柴尔德·凯撒利亚基金会的阿丽亚娜·德罗斯柴尔德夫人博士计划。

注：图1中介绍了完整的合成细胞生产协议的图示。根据用户的需求，协议的蛋白质表达（第3.2节）和合成细胞形成（第4节）部分也可以独立进行（进行一些调整）。
1. 制备S30-T7莱沙
<ol><li>条纹板大肠杆菌BL21（DE3）细菌转化与T7RNA聚合酶表达pAR1219质粒在LB-agar板补充50微克/mL安培霉素，以获得单菌落。</li>
	<li>准备启动溶液：将单个菌体接种成5 mL...

<ol>
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该协议引入了一种简单且经济的方法，用于生产大量产生蛋白质的合成细胞。活性细胞的产生取决于谨慎和准确地执行协议，并侧重于几个关键步骤。在该方法的精致部分，在细胞水致之前达到适当的细菌密度，在细菌水致酶中达到足够数量的蛋白质至关重要。其次，在4°C下进行流化过程，用液氮快速冷冻液化酶，以保持蛋白质活性。此外，在该协议中，我们使用大肠杆菌BL21（DE3）细胞?...

合成细胞是类似细胞的人造粒子，模仿活细胞的一个或多个功能，如分裂、形成膜相互作用和根据遗传代码11、2、32,3合成蛋白质的能力。包含无细胞蛋白合成（CFPS）系统的合成细胞具有很高的模块化，因为它们能够在DNA模板发生改变后产生各种蛋白质和RNA序列。CFPS系统以细胞裂解、纯化成分或合成成分为基础，提供一种有吸引力的替代蛋白，包括蛋白质合成所需的所有转录和翻译机制，如核糖体、RNA聚合酶、氨基酸和能量源（例如，三磷甘油酸盐和三磷酸腺）4、5、6、7、8、9。4,5,6,7,8,9将CFPS系统封装在脂质囊泡内，无需依赖活细胞10即可简单高效地生产蛋白质。此外，该平台允许合成可能在自然细胞内降解的肽，产生对活细胞有毒的蛋白质，以及用非天然氨基酸11，12,12修饰蛋白质。合成细胞已被作为研究目的的模型，研究从进化角度1，13,13实现细胞寿命所需的最小细胞成分。合成细胞也被用来建立和实施遗传回路，并作为定向进化的模型14，15，16。15,1614其他研究的重点是合成细胞模仿自然细胞生物活动的能力，旨在取代受损的自然细胞，如糖尿病患者的β细胞。此外，这些CFPS封装合成细胞产生各种治疗性蛋白质的能力说明了其潜力，被纳入临床使用18。
在这里，我们描述了一个自下而上的实验室规模协议（图1），用于生产基于CFPS系统封装在脂质囊泡中的RNA和产生蛋白质的合成细胞。这表明，合成细胞平台作为新型药物输送系统，用于在体内生产治疗性蛋白质药物的可能性。以前的研究已经研究了CFPS反应和细胞水合制剂过程44，8，208,20的优化。此外，还应用了几种细胞大小的脂质体制备技术，如微流体和聚合物基液滴稳定方法21、22、23，,22,在脂质体脂质组成24、25、2625,26中也有所不同24。23在所介绍的协议中，合成细胞采用水浸液转移法产生，封装过程在低温（<4°C）5、10、24、27、285,10,24,27,28下进行。这些温和的条件被发现有利于保持分子机械的生物功能完整性，即核糖体和蛋白质27，29，30。27,29,30颗粒的脂质组成由胆固醇和1-棕榈-2-奥莱基-sn-糖-3-磷脂素（POPC）组成。第一种存在于所有哺乳动物细胞膜中，对膜的稳定性、刚度和渗透性降低至关重要，后者模仿哺乳动物磷脂成分11、13。11,细胞转录和转化分子机械从BL21（DE3）大肠杆菌（大肠杆菌）菌株中提取，该菌株由pAR1219质粒过度表达T7RNA聚合酶转化，以增加CFPS效力和蛋白质合成。该系统已用于产生诊断和治疗蛋白，31体外和体内19的分子量高达66kDa。以下协议为合成细胞系统的生产提供了一种简单而有效的方法，可以解决与自然界中蛋白质合成相关的广泛基本问题，也可用于药物输送应用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td colspan="4" >A. Reagents required for step 1 (S30-T7 lysate preparation)</td>
		</tr>
		<tr >
			<td >E.coli BL21 (DE3)&#160;</td>
			<td >NEB</td>
			<td >C2527</td>
			<td >E.coli BL21 (DE3).</td>
		</tr>
		<tr >
			<td >pAR1219</td>
			<td >Sigma</td>
			<td >T2076</td>
			<td >TargeTron vector for transformation.</td>
		</tr>
		<tr >
			<td >Stock solution of 50 mg/mL Ampicillin</td>
			<td >Sigma</td>
			<td >A9518</td>
			<td >Stored at -20 &#176;C.</td>
		</tr>
		<tr >
			<td >10 g/L Bacto-tryptone</td>
			<td >BD Bioscience&#160;</td>
			<td >211705</td>
			<td rowspan="5" >For preparation of Luria Bertani (LB) agar (1.5%) plate.</td>
		</tr>
		<tr >
			<td >10 g/L Sodium chloride (NaCl)</td>
			<td >Bio-Lab</td>
			<td>19030591</td>
		</tr>
		<tr >
			<td >5 g/L Bacto-Yeast extract</td>
			<td >BD Bioscience&#160;</td>
			<td >212750</td>
		</tr>
		<tr >
			<td >15 g/L Agar agar purified</td>
			<td >Merck</td>
			<td>1.01614.5007</td>
		</tr>
		<tr >
			<td >50 &#181;g/mL Ampicillin</td>
			<td >Sigma</td>
			<td >A9518</td>
		</tr>
		<tr >
			<td >10 g/L Bacto-tryptone</td>
			<td >BD Bioscience&#160;</td>
			<td >211705</td>
			<td rowspan="4" >For preparation of Luria Bertani (LB) media (20 mL).</td>
		</tr>
		<tr >
			<td >10 g/L Sodium chloride (NaCl)</td>
			<td >Bio-Lab</td>
			<td>19030591</td>
		</tr>
		<tr >
			<td >5 g/L Bacto-Yeast extract</td>
			<td >BD Bioscience&#160;</td>
			<td >212750</td>
		</tr>
		<tr >
			<td >50 &#181;g/mL Ampicillin</td>
			<td >Sigma</td>
			<td >A9518</td>
		</tr>
		<tr >
			<td >12 g/L Bacto-tryptone</td>
			<td >BD Bioscience&#160;</td>
			<td >211705</td>
			<td rowspan="6" >For preparation of Terrific Broth (TB) media (1 L).</td>
		</tr>
		<tr >
			<td >24 g/L Bacto-Yeast extract</td>
			<td >BD Bioscience&#160;</td>
			<td >212750</td>
		</tr>
		<tr >
			<td >4% (v/v) Glycerol anhydrous</td>
			<td >Bio-Lab</td>
			<td >7120501</td>
		</tr>
		<tr >
			<td >2.32 g/L K2HPO4</td>
			<td>Spectrum chemical</td>
			<td>P1383</td>
		</tr>
		<tr >
			<td >12.54 g/L KH2PO4</td>
			<td>Spectrum chemical</td>
			<td>P1380</td>
		</tr>
		<tr >
			<td >&#160;50 &#181;g/mL Ampicillin</td>
			<td >Sigma</td>
			<td >A9518</td>
		</tr>
		<tr >
			<td >Stock solution of 100 mM Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)&#160;</td>
			<td >INALCO</td>
			<td >INA-1758-1400</td>
			<td >Filtered using 0.2 &#181;m hydrophilic PVDF syringe filter.</td>
		</tr>
		<tr >
			<td >Stock solution of 0.1 M dithiothreitol (DTT)&#160;</td>
			<td >TCI</td>
			<td >D1071</td>
			<td >Filtered using 0.2 &#181;m hydrophilic PVDF syringe filter.</td>
		</tr>
		<tr >
			<td >10 mM Tris-acetate at pH = 7.4</td>
			<td >Sigma</td>
			<td >T1503</td>
			<td rowspan="5" >S30 lysate buffer (1.5 L)</td>
		</tr>
		<tr >
			<td >14 mM magnesium acetate</td>
			<td >Merck</td>
			<td >1.05819.0250</td>
		</tr>
		<tr >
			<td >60 mM potassium acetate</td>
			<td >Carlo Erba</td>
			<td >470147</td>
		</tr>
		<tr >
			<td >1 mM DTT</td>
			<td >TCI</td>
			<td >D1071</td>
		</tr>
		<tr >
			<td >0.5 mL/L 2-mercaptoethanol</td>
			<td >Sigma</td>
			<td >M6250</td>
		</tr>
		<tr >
			<td colspan="4" >Equipment required for step 1</td>
		</tr>
		<tr >
			<td >100 mL sterilized Erlenmeyer flasks</td>
			<td >Thermo Scientific</td>
			<td >50-154-2846</td>
			<td >2 flasks</td>
		</tr>
		<tr >
			<td >2 L sterilized Erlenmeyer flasks with baffles</td>
			<td >KIMAX-KIMBLE</td>
			<td >25630</td>
			<td >2 flasks</td>
		</tr>
		<tr >
			<td >Floor incubator shaker</td>
			<td >MRC</td>
			<td >TOU-120-2</td>
			<td >Laboratory shaker incubator 450x450mm, 400rpm, 70 &#176;C</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Thermo Scientific</td>
			<td >75004270</td>
			<td >(75003340) - Fiberlite F10-6 x 100 LEX Fixed-Angle Rotor. 
			Should enable at least 13,000 x g.&#160;&#160;&#160;&#160; * Pre-cooled to 4 &#176;C.</td>
		</tr>
		<tr >
			<td >High pressure homogenizer</td>
			<td >AVESTIN</td>
			<td>EmulsiFlex-C3</td>
			<td >Pre-cooled to 4 &#176;C.</td>
		</tr>
		<tr >
			<td >-80oC freezer</td>
			<td>SO-LOW</td>
			<td>U85-18</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized 1.5 mL plastic tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td >Preferably pre-cooled to -20 &#176;C.</td>
		</tr>
		<tr >
			<td >Spectrophotometer</td>
			<td >TECAN</td>
			<td >IN-MNANO</td>
			<td >Infinite M200 pro</td>
		</tr>
		<tr >
			<td >96-well transparent plate</td>
			<td >Thermo Scientific</td>
			<td>167008</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized graduated cylinder</td>
			<td >Corning</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized centrifuge tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td >Preferably pre-cooled to -20 &#176;C.</td>
		</tr>
		<tr >
			<td >Sterilized pipette tips</td>
			<td >Corning</td>
			<td ></td>
			<td >Preferably pre-cooled to -20 &#176;C.</td>
		</tr>
		<tr >
			<td >Crushed ice bucket</td>
			<td>Bel-Art</td>
			<td >M18848-4001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Small liquid nitrogen tank</td>
			<td>NALGENE</td>
			<td>4150-4000</td>
			<td ></td>
		</tr>
		<tr >
			<td colspan="4" >B. Reagents required for step 3 (lipids in oil solution preparation):</td>
		</tr>
		<tr >
			<td >1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td>
			<td >Lipoid</td>
			<td >556400</td>
			<td >Powder</td>
		</tr>
		<tr >
			<td >Cholesterol</td>
			<td >Sigma</td>
			<td >C8667</td>
			<td >Powder</td>
		</tr>
		<tr >
			<td >Chloroform</td>
			<td >Bio-Lab</td>
			<td >3082301</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mineral oil</td>
			<td >Sigma</td>
			<td >M5904</td>
			<td >Light oil</td>
		</tr>
		<tr >
			<td colspan="4" >Equipment required for step 2</td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td >Scientific industries</td>
			<td >SI-0256</td>
			<td ></td>
		</tr>
		<tr >
			<td >Heating block</td>
			<td >TECHNE</td>
			<td >FDB03AD</td>
			<td >Pre-heated to 80 &#176;C. 
			Should enable controlled temperature.&#160;</td>
		</tr>
		<tr >
			<td >2 mL screw neck glass vials</td>
			<td >CSI Analytical Innovations</td>
			<td >VT009M-1232</td>
			<td rowspan="2" >For a larger scale, use 50 mL falcons and evaporate the chloroform using rotary evaporator.</td>
		</tr>
		<tr >
			<td >9mm Screw Cap</td>
			<td >CSI Analytical Innovations</td>
			<td >C395R-09LC</td>
		</tr>
		<tr >
			<td colspan="4" >C. Reagents required for step 3 (inner and feeding reaction mixtures):</td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td >Spectrum</td>
			<td >H1089</td>
			<td rowspan="2" >1 M HEPES-KOH (pH = 8) - pH buffer</td>
		</tr>
		<tr >
			<td >Potassium hydroxide (KOH)</td>
			<td >Frutarom</td>
			<td >55290</td>
		</tr>
		<tr >
			<td >1 M Magnesium acetate</td>
			<td >Merck</td>
			<td >1.05819.0250</td>
			<td >Co-factor and negative charge stabilizor.</td>
		</tr>
		<tr >
			<td >1 M Potassium acetate</td>
			<td >Carlo Erba</td>
			<td >470147</td>
			<td >Negative charge stabilizor.</td>
		</tr>
		<tr >
			<td >5.2 M Ammonium acetate</td>
			<td >Merck</td>
			<td >1.01116.1000</td>
			<td >Stabilizes negative charge.</td>
		</tr>
		<tr >
			<td >50% (w/v) Polyethylene glycol 6000 (PEG)</td>
			<td >Merck</td>
			<td >8.07491.1000</td>
			<td >Increases the concentration of the macromolecules.</td>
		</tr>
		<tr >
			<td >0.5 M 3-phosphoglycerate (3-PGA)</td>
			<td>Sigma</td>
			<td>P8877</td>
			<td >Secondary energy source.</td>
		</tr>
		<tr >
			<td >50 mM Amino acids mixture I</td>
			<td>Sigma</td>
			<td>LAA21-1KT</td>
			<td >Amino acids additive. 
			Contains: 50 mM of each of the following 17&#160; natural amino acids - alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, and valine.</td>
		</tr>
		<tr >
			<td >50 mM Amino acids mixture II</td>
			<td>Sigma</td>
			<td>LAA21-1KT</td>
			<td >Amino acids additive. 
			Contains: 50 mM of each of the following 3 natural amino acids - tryptophan, phenylalanine, and tyrosine.</td>
		</tr>
		<tr >
			<td >100 mM Adenine triphosphate (ATP)</td>
			<td>Sigma</td>
			<td>A3377</td>
			<td >Nucleotides &#38; energy source.</td>
		</tr>
		<tr >
			<td >50 mM Guanidine triphosphate (GTP)</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td >Nucleotides &#38; energy source.</td>
		</tr>
		<tr >
			<td >100 mM Uridine triphosphate (UTP)</td>
			<td>ACROS ORGANICS</td>
			<td>226310010</td>
			<td >Nucleotides additive.</td>
		</tr>
		<tr >
			<td >100 mM Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td >INALCO</td>
			<td >INA-1758-1400</td>
			<td >Genes expression induction.</td>
		</tr>
		<tr >
			<td >2 M Sucrose</td>
			<td >J.T. Baker</td>
			<td >1933078</td>
			<td >Generating a density gradient.</td>
		</tr>
		<tr >
			<td >2 M Glucose</td>
			<td >Sigma</td>
			<td >16301</td>
			<td >Generating a density gradient.</td>
		</tr>
		<tr >
			<td >H2O UltraPure Water (UPW)</td>
			<td >Bio-Lab</td>
			<td >2321777500</td>
			<td >DNase &#38; RNase free</td>
		</tr>
		<tr >
			<td >S30-T7 lysate</td>
			<td >_</td>
			<td >_</td>
			<td >Prepared at step 1.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Source of transcription &#38; translation components. 
			Store at -80 &#176;c, thaw on crashed ice just before usage.&#160;</td>
		</tr>
		<tr >
			<td >Stock of DNA plasmid of choice</td>
			<td >_</td>
			<td >_</td>
			<td >Contains the sequence for the requested protein. 
			Under T7 promotor</td>
		</tr>
		<tr >
			<td colspan="4" >D. Equipment required for step 4 (synthetic cells preparation)</td>
		</tr>
		<tr >
			<td rowspan="2" >Floor incubator shaker or Thermomixer</td>
			<td >MRC</td>
			<td >TOU-120-2</td>
			<td >Laboratory shaker incubator 450x450mm, 400rpm, 70 &#176;C</td>
		</tr>
		<tr >
			<td >PHMT Grant Bio&#160;</td>
			<td >PSC18</td>
			<td >Thermomixer</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Thermo Scientific</td>
			<td >75004270</td>
			<td >(75003629) - TX-400 4 x 400mL Swinging Bucket Rotor. 
			Suited for 15 mL sized tubes. 
			Preferably swinging buckets. 
			Should enable at least 1000 x g. 
			Pre-cooled to 4 &#176;C.</td>
		</tr>
		<tr >
			<td >Table centrifuge</td>
			<td >Thermo Scientific</td>
			<td >75002420</td>
			<td >(75003424) - 24 x 1.5/2.0mL rotor with ClickSeal. 
			Suited for Eppendorf vials. 
			Pre-cooled to 4 &#176;C.</td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td >Scientific industries</td>
			<td >SI-0256</td>
			<td ></td>
		</tr>
		<tr >
			<td >Crushed ice bucket</td>
			<td >Bel-Art</td>
			<td >M18848-4001</td>
			<td ></td>
		</tr>
		<tr >
			<td >2 mL screw neck glass vials</td>
			<td >CSI Analytical Innovations</td>
			<td >VT009M-1232</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterile 15 mL plastic tubes</td>
			<td >Thermo Scientific</td>
			<td >339651</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized 1.5 mL plastic tubes</td>
			<td >Eppendorf</td>
			<td >30120086</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sterilized pipette tips</td>
			<td >Corning</td>
			<td ></td>
			<td >Sterilized by autoclave.</td>
		</tr>
	</tbody>
</table>

preparing protein producing synthetic cells using cell free bacterial extracts, liposomes and emulsion transfer

合成细胞构造的自下而上装配方法是隔离和研究细胞模拟环境中细胞过程的有效工具。此外，无细胞表达系统的发展已经证明了以受控方式重组蛋白质生产、转录和翻译过程（DNA+RNA+蛋白质）的能力，利用合成生物学。在这里，我们描述了一种制备无细胞表达系统的协议，包括生产一种有效的细菌水合物，并将这种水泡封装在富含胆固醇的脂质型巨型单层囊泡（GUVs）（即稳定脂质体）内，以形成合成细胞。该协议描述了合成细胞成分的准备方法，包括活性细菌裂解物的产生，然后根据油水乳液转移方法对合成细胞进行详细的逐步制备。这些有助于以简单和负担得起的方式生产数百万个合成细胞，具有用于生产不同类型的蛋白质的高多功能性。所得合成细胞可用于研究分离环境中的蛋白质/RNA的产生和活性，在定向进化中，以及作为在体内按需生产治疗蛋白的受控药物输送平台。

我们提出了一种合成细胞制备协议，通过封装基于BL21大肠杆菌体内脂质囊泡的S30-T7 CFPS系统。图 2中介绍了准备过程的原理图描述，其中包括每个阶段的图像。合成细胞制备过程的成功取决于每个阶段的适当性能，并受不同参数的影响。应调整协议以适应特定蛋白质的产生。
在CFPS散量反应和合成细胞中，将表达模型蛋白超级文件夹绿色荧光蛋白?...

Watch this Scientific Journal Video about Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer at JoVE.com

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

该协议描述了用于自下而上制备RNA和产生合成细胞的蛋白质的方法、材料、设备和步骤。合成细胞的内水腔含有S30细菌裂解，封装在脂质双层层（即稳定脂质体）内，使用油水乳液转移方法。

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

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

蛋白质生产合成细胞提供了一个多功能的平台，用于研究生命的起源以及基于细胞的合成疗法，用于高级药物的输送。该方法简单且价格实惠，适用于RNA和蛋白质表达，可直接应用于体内的现场治疗蛋白生产。这些系统可用于治疗各种疾病，从代谢疾病和蛋白质替代到癌症和糖尿病。此多步骤协议具有定义的停止点。首次执行时，建议将工作分成几天。合成细胞生产的协议始于无细胞S30-T7酸盐的制备。接下来，膜脂质和细胞营养液准备模拟内部环境，并支持蛋白质生产。最后一步是合成细胞本身的准备。在 100 毫升 Erlenmeyer 烧瓶中准备重复的开胃培养物。在每个烧瓶中，添加5毫升LB介质，每毫升安皮西林补充50微克，并接种单个大肠杆菌菌群。在 250 rpm 和 37 摄氏度的地板孵化器摇床中过夜地种植这种文化。接下来，准备两升困惑的 Erlenmeyer 烧瓶，含有 500 毫升的 TB 介质，并辅以每毫升安皮西林 50 微克。接种每两升烧瓶与五毫升的开胃培养。在 250 rpm 和 37 摄氏度的转速下将烧瓶放在摇床中。使用分光光度计定期监测培养物，并在 0.8 和 1 之间将培养至 OD600。要诱导T7RNA聚合酶表达，在每个烧瓶中加入三毫升100毫摩尔IPTG，达到0.6毫摩尔。继续种植培养，直到 OD600 大约四个。培养物达到所需的光学密度后，将悬浮液从每个 Erlenmeyer 烧瓶转移到两个 250 毫升灭菌离心管中。在摄氏4度下，以7000倍g离心管，10分钟。丢弃上一杯。如果需要，使用搅拌器将每个颗粒在 250 毫升的冷 S30 lysate 缓冲液中重新使用。在摄氏4度下以7000倍g离心10分钟。丢弃上清液，将所有颗粒重新在 15 毫升冷 S30 莱盐缓冲液中重新暂停。使用纱布垫过滤悬架。在继续下一步之前，先将储存 lysate 所需的 1.5 毫升小瓶中的提示预冷。两次均匀化悬浮液。使用工作压力为 15，000 PSI，空气压力为 4 bar，可进行电池破裂。收集同质。每10毫升均质悬浮液中加入100微升0.1摩尔DTT。在4摄氏度下30分钟将悬浮液在24，700次g下离心。DTT 和氯仿被归类为刺激物和有害物，因此应谨慎处理。稍后在协议中使用的氯仿必须在具有烟气提取的区域使用。要保留 lysate 活动，请快速执行该步骤。将200微升上清液等同物放在预冷却小瓶中，并立即用液氮捕捉冷冻。将小瓶存放在负 80 摄氏度，供将来使用。将氯仿中单独溶解波普克和胆固醇，最终浓度为每毫升100毫克。分别旋转每个小瓶。将组件组合在两毫升玻璃瓶中。加入50微升的胆固醇-氯仿溶液、50微升的波普克-氯仿溶液和500微升的矿物油。漩涡小瓶，然后蒸发氯仿，在80摄氏度的化学罩中加热约一小时。在开始合成细胞生产阶段之前，请准备手稿中描述的外层、预内和进料解决方案。然后将1.2毫升的外层溶液放在15毫升的管子中，然后慢慢从第一个玻璃瓶中加入一层500微升的脂质油溶液。在室温下孵育管子20分钟。要完成内部溶液制备，通过添加S30-T7立质和DNA质粒，将冰混合到100微升的最终体积。将 100 微升内溶液与立盐和质粒添加到第二个两毫升玻璃瓶中，并加入 500 微升脂油溶液。管道上下大力一分钟，然后漩涡在五级再旋一分钟。在碎冰上孵育所得乳液10分钟后，在15毫升管中缓慢地将乳液加入油相顶部。在100倍的g和4摄氏度下将管子离心10分钟。然后在400倍的g和4摄氏度下离心10分钟。离心结束时，在管的底部应能观察到颗粒。要提取颗粒，请先清除多余的油层。接下来，用大约 400 微升的外部溶液加载经过修剪的移液器尖端，并使用移液器尖端收集颗粒，当移液器尖端通过油相时释放外部溶液。擦拭移液器尖端，将颗粒转移到干净的 1.5 毫升管中。在1000倍的g和4摄氏度下将颗粒离心10分钟。取出上一杯，将颗粒重新在100微升的进料溶液中。对于蛋白质表达，在37摄氏度下孵育悬浮液两小时，不摇晃。表达模型蛋白sfGFP和雷尼拉荧光素酶的质粒被引入无细胞蛋白合成、CFPS体积反应和合成细胞中。使用几种方法对蛋白质生产进行评估。西方印迹分析检测到cfPS散装反应和合成细胞内的sfGFP His6生产。纯化 sfGFP His6 用作正对控件。带光明场和 GFP 过滤器的荧光显微镜显示产生 sfGFP 的合成细胞。流细胞学用于分析产生合成细胞的sfGFP。活性合成细胞量的计算基于由红色直方图表示的负DNA样本定义的sfGFP荧光强度阈值。从活性合成细胞组中计算出产生合成细胞的sfGFP平均荧光强度，并规范化为阴性DNA样本。根据sfGFP信号计算活性合成细胞的直径分布。用发光测量来量化雷尼拉荧光素酶活性。该方法用途广泛，可以通过改变利盐原产、脂质成分和内部溶液浓度，针对不同的靶蛋白进行优化。可以对产生荧光标记蛋白的合成细胞进行FACS分析，以提供活性合成细胞分数的定量值。西方的印迹分析将测量蛋白质的产生。

Research

JoVE Journal

Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

科研

生物工程

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

人体软组织的生物力学特性采用压痕和拉伸试验

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

利用合成生物学工程师活细胞接口，具有可编程材料

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

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

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

快速生长的纳氏弧菌的无细胞蛋白表达

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

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...