Name: in vesiculo synthesis of peptide membrane precursors for autonomous vesicle growth
Uploaded: 2019-06-28T14:10:00
Description: Watch this Scientific Journal Video about In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth at JoVE.com

我们感谢通过DFG TRR 235(生命出现,项目P15)、欧洲研究理事会(赠款协议694410 AEDNA)和TUM国际科学与工程研究生院IGSSE(项目9.05)提供的财政支持。.我们感谢E.Falgenhauer在样品制备方面的帮助。我们感谢A.杜平和M.施瓦茨-席林在TX-TL系统和有益的讨论方面所做的帮助。我们感谢荷兰进行了有益的讨论。

1. 类似艾莱他一样的多肽的表达
<ol><li>第1天:为肽表达准备启动培养和供应
	<ol>
<li>制备和高压灭菌表达培养瓶(4 x 2.5 L)和3升LB介质。对于 1 L LB 介质,将 25 g LB 粉末添加到 1 L 的超纯水中。</li>
		<li>使用 100 mL LB 培养基、50 μL 无菌过滤 (0.22 μm 过滤器) 氯霉素溶液(EtOH 中为 25 mg/mL)和 50 μL 无菌过滤 (0.22 μm 过滤器) 卡比霉素溶液(50% EtOH 和 50% 超纯水)制备起动机培养基。</li>
		<li>从含有...

<ol>
	<li>Chen, I. A., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+kinetic+study+of+the+growth+of+fatty+acid+vesicles.">A kinetic study of the growth of fatty acid vesicles.</a> Biophysical Journal. 87 (2), 988-998 (2004).</li><li>Nourian, Z., Roelofsen, W., Danelon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggered+gene+expression+in+fed-vesicle+microreactors+with+a+multifunctional+membrane.">Triggered gene expression in fed-vesicle microreactors with a multifunctional membrane.</a> Angewandte Chemie International Edition. 51 (13), 3114-3118 (2012).</li><li>Hardy, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-reproducing+catalyst+drives+repeated+phospholipid+synthesis+and+membrane+growth.">Self-reproducing catalyst drives repeated phospholipid synthesis and membrane growth.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (27), 8187-8192 (2015).</li><li>Scott, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Free+Phospholipid+Biosynthesis+by+Gene-Encoded+Enzymes+Reconstituted+in+Liposomes.">Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes.</a> PLoS ONE. 11 (10), e0163058 (2016).</li><li>Deamer, D. W., Barchfeld, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+macromolecules+by+lipid+vesicles+under+simulated+prebiotic+conditions.">Encapsulation of macromolecules by lipid vesicles under simulated prebiotic conditions.</a> Journal of Molecular Evolution. 18 (3), 203-206 (1982).</li><li>Adamala, K., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonenzymatic+Template-Directed+RNA+Synthesis+Inside+Model+Protocells.">Nonenzymatic Template-Directed RNA Synthesis Inside Model Protocells.</a> Science. 342 (6162), 1098-1100 (2013).</li><li>Zhu, T. F., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupled+growth+and+division+of+model+protocell+membranes.">Coupled growth and division of model protocell membranes.</a> Journal of the American Chemical Society. 131 (15), 5705-5713 (2009).</li><li>Kurihara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-reproduction+of+supramolecular+giant+vesicles+combined+with+the+amplification+of+encapsulated+DNA.">Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA.</a> Nature Chemistry. 3 (10), 775-781 (2011).</li><li>Chu, H. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+analysis+of+an+elastin-like+polypeptide+(ELP)+in+a+cell-free+protein+synthesis+system.">Expression analysis of an elastin-like polypeptide (ELP) in a cell-free protein synthesis system.</a> Enzyme and Microbial Technology. 46 (2), 87-91 (2010).</li><li>Martino, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Expression,+Aggregation,+and+Triggered+Release+from+Polymersomes+as+Artificial+Cell-like+Structures.">Protein Expression, Aggregation, and Triggered Release from Polymersomes as Artificial Cell-like Structures.</a> Angewandte Chemie International Edition. 51 (26), 6416-6420 (2012).</li><li>Vogele, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+synthetic+cells+using+peptide-based+reaction+compartments.">Towards synthetic cells using peptide-based reaction compartments.</a> Nature Communications. 9 (1), 3862 (2018).</li><li>Huber, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+amphiphilic+proteins+as+building+blocks+for+the+intracellular+formation+of+organelle-like+compartments.">Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments.</a> Nature Materials. 14 (1), 125-132 (2014).</li><li>McPherson, D. T., Xu, J., Urry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product+purification+by+reversible+phase+transition+following+Escherichia+coli+expression+of+genes+encoding+up+to+251+repeats+of+the+elastomeric+pentapeptide+GVGVP.">Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP.</a> Protein Expression and Purification. 7 (1), 51-57 (1996).</li><li>Urry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+chemistry+of+biological+free+energy+transduction+as+demonstrated+by+elastic+protein-based+polymers.">Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers.</a> The Journal of Physical Chemistry B. 101 (51), 11007-11028 (1997).</li><li>Urry, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin:+a+representative+ideal+protein+elastomer.">Elastin: a representative ideal protein elastomer.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 357 (1418), 169-184 (2002).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+recombinant+proteins+by+fusion+with+thermally-responsive+polypeptides.">Purification of recombinant proteins by fusion with thermally-responsive polypeptides.</a> Nature Biotechnology. 17 (11), 1112-1115 (1999).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+synthesis+of+protein-based+polymers+with+precisely+specified+molecular+weight+and+sequence+by+recursive+directional+ligation:+examples+from+the+elastin-like+polypeptide+system.">Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.</a> Biomacromolecules. 3 (2), 357-367 (2002).</li><li>Sun, Z. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+implementing+an+Escherichia+coli+based+TX-TL+cell-free+expression+system+for+synthetic+biology.">Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.</a> Journal of Visualized Experiments. (79), e50762 (2013).</li><li>Caschera, F., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+2.3+mg/ml+of+protein+with+an+all+Escherichia+coli+cell-free+transcription-translation+system.">Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system.</a> Biochimie. 99, 162-168 (2014).</li><li>Schwarz-Schilling, M., Aufinger, L., M&#252;ckl, A., Simmel, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+communication+between+bacteria+and+cell-free+gene+expression+systems+within+linear+chains+of+emulsion+droplets.">Chemical communication between bacteria and cell-free gene expression systems within linear chains of emulsion droplets.</a> Integrative Biology. 8 (4), 564-570 (2016).</li><li>Garamella, J., Marshall, R., Rustad, M., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+All+E.+coli+TX-TL+Toolbox+2.0:+A+Platform+for+Cell-Free+Synthetic+Biology.">The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.</a> ACS Synthetic Biology. 5 (4), 344-355 (2016).</li><li>Rideau, E., Dimova, R., Schwille, P., Wurm, F. R., Landfester, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+and+polymersomes:+a+comparative+review+towards+cell+mimicking.">Liposomes and polymersomes: a comparative review towards cell mimicking.</a> Chemical Society Reviews. 47 (23), 8572-8610 (2018).</li><li>Filonov, G. S., Moon, J. D., Svensen, N., Broccoli Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broccoli:+Rapid+Selection+of+an+RNA+Mimic+of+Green+Fluorescent+Protein+by+Fluorescence-Based+Selection+and+Directed+Evolution.">Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution.</a> Journal of the American Chemical Society. 136 (46), 16299-16308 (2014).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+Plasmid+DNA+from+Bacterial+Colonies+Using+the+Qiagen+Miniprep+Kit.">Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit.</a> Journal of Visualized Experiments. (6), 247 (2007).</li><li>Stetefeld, J., McKenna, S. A., Patel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+light+scattering:+a+practical+guide+and+applications+in+biomedical+sciences.">Dynamic light scattering: a practical guide and applications in biomedical sciences.</a> Biophysical Reviews. 8 (4), 409-427 (2016).</li></ol>

薄膜补液是创建小型单片囊泡的常见程序。故障的主要来源是对程序中使用的材料处理错误。

最初,ELPs由大肠杆菌细胞产生。ELP 纯化后的产量可能有很大差异,具体取决于协议在关键步骤中执行的谨慎程度。这些是逆温循环 (ITC) 步骤和降水后 ELP 的重新悬浮。为了纯化,我们通过添加磷酸触发了肽的疏水性崩溃,磷酸将pH?...

合成活细胞系统的创建通常从两个不同的方向进行。在自上而下的方法中,细菌的基因组被简化为其基本成分,最终导致最小的细胞。在自下而上的方法中,人工细胞从分子组分或细胞子系统中组装,需要功能上集成到一致的细胞样系统中。
在de novo方法中,必要的生化成分的分门别类通常是使用磷脂或脂肪酸1、2、3、4制成的膜实现的。这是因为"现代"细胞膜主要由磷脂组成,而脂肪酸被认为是益生菌膜外壳5、6的可信候选体。为了形成新膜或促进膜生长,必须从外部7提供两栖积木,或者最好使用相应的合成代谢工艺在膜腔内生产4 ,8.
脂质合成是一个相对复杂的代谢过程,而肽可以很容易地使用无细胞基因表达反应9,10。因此,由两栖肽形成的肽膜是脂质膜的有趣替代品,作为能够生长11的人工细胞模拟的外壳。
嗜血弹性蛋白样二块共聚物(ELPs)是一种有吸引力的肽类,它可以作为这种膜12的构建基块。ELPs的基本氨基酸序列图案是(GaGVP)n,其中"a"可以是除proline以外的任何氨基酸,"n"是主题重复的数量13,14,15,16,17.ELPs已经创建与主要含有苯丙氨酸a和亲水性块主要由谷氨酸11组成的疏水块。根据和溶液参数(如pH和盐浓度),ELPs在温度Tt下表现出所谓的逆温过渡,其中肽经历了从亲水性到疏水性完全可逆的相变状态。使用"TX-TL"细菌细胞提取物11、18、19、20、21,在囊泡内轻松实现肽的合成,这提供了耦合转录和翻译反应所需的所有组件。
TX-TL系统封装在一起,DNA模板利用玻璃珠的脱水补液将ELP编码到ELP囊泡中作为固体支撑。囊泡的形成是通过从珠表面11的干肽的补液而形成的。可以使用其他方法22用于囊泡形成,这可能显示较低的多分散度和较大的囊泡尺寸(例如,电形成、乳液相转移或基于微流体的方法)。为了测试封装方法的可行性,荧光能的转录法dBroccoli2可以交替使用11,这比TX-TL系统的基因表达要复杂。
由于囊泡中膜积块的表达及其随后的加入膜,囊泡开始生长11。ELP的膜结合可以通过FRET测定进行演示。为此,用于形成初始囊泡总体的 ELPs 与构成 FRET 对的相等份额中的荧光染料结合使用。当在囊泡中表达未标记的ELP并将其并融入膜中时,膜中标记的ELP被稀释,因此FRET信号减小11。铜催化阿齐德-烷基环增法是一种通用且常见的结合方法。使用稳定配体,如三s(苯基苯甲酰乙-胺),反应可以在生理pH的水溶液中进行,无需水解反应物11,这适用于结合反应涉及肽。
以下协议详细介绍了生长基于ELP的肽类的制备情况。介绍了使用玻璃珠法的肽和囊泡形成表达式。此外,还介绍了如何实现氟化德布罗图阿图默的转录和ELP囊泡内蛋白质表达的转录-翻译反应。最后,提供了一个ELPs与荧光荧光的结合程序,它可以用来通过FRET测定11来证明囊泡的生长。

<table>
	<tbody>
		<tr>
			<td>2xYT</td>
			<td>MP biomedicals</td>
			<td>3012-032</td>
			<td></td>
		</tr>
		<tr>
			<td>3-PGA</td>
			<td>Sigma-Aldrich</td>
			<td>P8877</td>
			<td></td>
		</tr>
		<tr>
			<td>5PRIME Phase Lock GelTM tube</td>
			<td>VWR</td>
			<td>733-2478</td>
			<td></td>
		</tr>
		<tr>
			<td>alkine-conjugated Cy3</td>
			<td>Sigma-Aldrich</td>
			<td>777331</td>
			<td></td>
		</tr>
		<tr>
			<td>alkine-conjugated Cy5</td>
			<td>Sigma-Aldrich</td>
			<td>777358</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A8937</td>
			<td></td>
		</tr>
		<tr>
			<td>benzamidin</td>
			<td>Carl Roth</td>
			<td>CN38.2</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 Rosetta 2 E. coli strain</td>
			<td>Novagen</td>
			<td>71402</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford BSA Protein Assay Kit</td>
			<td>Bio-rad</td>
			<td>500-0201</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma-Aldrich</td>
			<td>A9501</td>
			<td></td>
		</tr>
		<tr>
			<td>carbenicillin</td>
			<td>Carl Roth</td>
			<td>6344.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C1919</td>
			<td></td>
		</tr>
		<tr>
			<td>chloramphenicol</td>
			<td>Carl Roth</td>
			<td>3886.3</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Carl Roth</td>
			<td>4432.1</td>
			<td></td>
		</tr>
		<tr>
			<td>CoA</td>
			<td>Sigma-Aldrich</td>
			<td>C4282</td>
			<td></td>
		</tr>
		<tr>
			<td>CTP</td>
			<td>USB</td>
			<td>14121</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>Carl Roth</td>
			<td>P024.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DFHBI</td>
			<td>Lucerna Technologies</td>
			<td>410</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Carl Roth</td>
			<td>A994.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>NEB</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carl Roth</td>
			<td>9065.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F7878</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads, acid-washed</td>
			<td>Sigma-Aldrich</td>
			<td>G1277</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>USB</td>
			<td>16800</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H6147</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG (&#946;-isopropyl thiogalactoside )</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>P017.1</td>
			<td></td>
		</tr>
		<tr>
			<td>K-glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1149</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Carl Roth</td>
			<td>X968.2</td>
			<td></td>
		</tr>
		<tr>
			<td>lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Carl Roth</td>
			<td>82.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>KK36.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg-glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>49605</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Bio-Spin Chromatography Columns</td>
			<td>Bio-Rad</td>
			<td>732-6204</td>
			<td></td>
		</tr>
		<tr>
			<td>NAD</td>
			<td>Sigma-Aldrich</td>
			<td>N6522</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS-azide linker (y-azidobutyric acid oxysuccinimide ester)</td>
			<td>Baseclick</td>
			<td>BCL-033-5</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>Carl Roth</td>
			<td>263.2</td>
			<td></td>
		</tr>
		<tr>
			<td>pH stripes</td>
			<td>Carl Roth</td>
			<td>549.2</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Carl Roth</td>
			<td>6367.2</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate-buffered saline</td>
			<td>VWR</td>
			<td>76180-684</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphoric acid</td>
			<td>Sigma-Aldrich</td>
			<td>W290017</td>
			<td></td>
		</tr>
		<tr>
			<td>polyethyleneimine</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic solution</td>
			<td>Sigma-Aldrich</td>
			<td>P8584</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic solution</td>
			<td>Sigma-Aldrich</td>
			<td>P8709</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAPol reaction buffer</td>
			<td>NEB</td>
			<td>B9012</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase inhibitor murine</td>
			<td>NEB</td>
			<td>M0314S</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap Wipes</td>
			<td>ThermoFisher</td>
			<td>AM9788</td>
			<td></td>
		</tr>
		<tr>
			<td>rNTP</td>
			<td>NEB</td>
			<td>N0466S</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Phenol/Chloroform/Isoamyl alcohol</td>
			<td>Carlroth</td>
			<td>A156.1</td>
			<td></td>
		</tr>
		<tr>
			<td>RTS Amino Acid Sampler</td>
			<td>5 Prime</td>
			<td>2401530</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer Dialysis Cassettes, 10k MWCO (Kit)</td>
			<td>Thermo-Scientific</td>
			<td>66382</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Carl Roth</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Carl Roth</td>
			<td>8655.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>85558</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile-filtered (0.22 &#181;m filter)</td>
			<td>Carl Roth</td>
			<td>XH76.1</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 polymerase</td>
			<td>NEB</td>
			<td>M0251S</td>
			<td></td>
		</tr>
		<tr>
			<td>TBTA (tris(benzyltriazolylmethyl)amine)</td>
			<td>Sigma-Aldrich</td>
			<td>678937</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP (tris(2-carboxyethyl)-phosphine hydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>C4706</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fischer</td>
			<td>BP1521</td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA (from E. coli)</td>
			<td>Roche Applied Science</td>
			<td>MRE600</td>
			<td></td>
		</tr>
		<tr>
			<td>UTP</td>
			<td>USB</td>
			<td>23160</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vesiculo synthesis of peptide membrane precursors for autonomous vesicle growth

生化反应的分块化是合成细胞的核心方面。为此,肽反应室是脂体或脂肪酸基囊泡的诱人替代品。在体囊外部或内部,肽可以很容易地表达并简化膜前体的合成。此处提供了一种基于两栖弹性蛋白样多肽 (ELP) 利用玻璃珠脱水补液创建直径为 ±200 nm 的囊泡的协议。还介绍了细菌ELP表达和通过逆温循环纯化的协议,以及它们与荧光染料的共价功能化。此外,本报告还描述了一种使RNA aptamer dBroccoli在ELP囊泡内转录的协议,作为生化反应的一个不太复杂的例子。最后,提供了一个方案,允许荧光蛋白和膜肽的囊泡表达,而后者的合成导致囊泡生长。

囊泡生产 图1显示了用不同膨胀溶液和玻璃珠法制备的囊泡的透射电子显微镜(TEM)图像(另见Vogele等人11)。对于图1A中的样本,只有PBS用作膨胀溶液,以证明囊泡的形成并确定其大小。当TX-TL被用作膨胀溶液时(图1B),囊泡也形成。进行了动态光散射(DLS)测量,表明玻璃珠法对囊泡形成有影响。<strong class="xfig...

Watch this Scientific Journal Video about In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth at JoVE.com

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

这里介绍了创建能够生长的基于肽的小型单片杆菌囊泡的协议。为了便于膜肽的囊泡生产,这些囊泡配备了转录翻译系统和肽编码质粒。

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

在我们的协议中，我们使用小玻璃珠的薄膜再水化，形成由肽构成的反应室。在实验中，我们受益于协议的简单性和鲁棒性。这种技术的主要优点是能够吸收甚至敏感的样品，如使用的粗细胞提取物。与有机溶剂的接触会显著影响样品。要开始此过程，请使用离心真空浓缩器将 ELP 溶液浓缩至 1.1 毫摩尔。将这种浓缩溶液的200微升与2至1个氯仿和甲醇混合物的1，250微升混合。漩涡溶液彻底混合。接下来，在十毫升圆形底部烧瓶中加入1.5克球形玻璃珠。将 ELP 和氯仿甲醇溶液加入圆形底部烧瓶中，轻轻摇动以混合。将烧瓶连接到旋转蒸发器。将转速调整至 150 RPM，将压力调节至 20，000 帕斯卡约 4 分钟，直到液体在室温下蒸发。由于可能导致沸腾延迟，使用旋转蒸发器时必须小心。在此之后，松散地包裹铝箔围绕圆底烧瓶的开口，以防止玻璃珠的损失，并放入干燥器至少一个小时的烧瓶，以确保剩余的氯仿和甲醇蒸发。对于单个实验，将 100 毫克肽覆盖的玻璃珠与 60 微升的膨胀溶液混合。在25摄氏度下孵育该样品5分钟。使用桌面离心机快速离心样品并沉淀玻璃珠。然后使用移液器收集上流水，其中含有囊泡。首先，将预纯质粒DNA的100微升与100微升的罗提-苯酚、氯仿或异氨醇混合在微离心管中，以便更好地进行相分离。轻轻反转管长达六次，在16，000倍g下离心，室温为5分钟。然后，在上相中加入200微升氯仿，并反转管子多达6次。将样品在16，000倍g下离心，室温为5分钟。在此之后，将上流液器移液到单独的管中，加入10微升的三摩尔醋酸钠进行乙醇沉淀。在80摄氏度下加入一毫升冷乙醇，在80摄氏度储存样品一小时。接下来，将样品在16，000倍g和4摄氏度下离心15分钟。去清液，在20摄氏度下加入一毫升冷70%乙醇。在16，000倍的g和4摄氏度的离心机5分钟。然后，通过移液去除液体，小心不要干扰DNA颗粒。将样品在室温下储存约15分钟，以蒸发剩余的乙醇。在此之后，在样品中加入超纯水，将样品浓度调整为约300纳米摩尔，以260纳米的吸收为单位进行测量。要准备转录-翻译反应，请遵循准备好的粗细胞提取和冰上反应缓冲液。对于60微升反应混合物，将血浆DNA加入反应缓冲液的37.5微升，加入28.7微升的粗细胞提取物，在58.8微升的最终体积中加入超纯水。在反应开始前，加入1.2微升的T7RNA聚合酶溶液，上下移液器混合。在实验期间，在29摄氏度下孵育样品和膨胀溶液，通常为4至8小时。囊泡的透射电子显微镜图像显示，各种膨胀溶液，如TX/TL，甚至只有PBS可用于形成囊泡。对于这两种解决方案，尺寸确定是一个直截了当的步骤。动态光散射表明，没有玻璃珠法的囊泡直径为134纳米，使用玻璃珠时多散度为25%，直径为168纳米，多散度为21%， 两种荧光蛋白的荧光强度在ELP囊内表达，然后用荧光板读取器进行测量。需要注意的是，囊泡形成后，囊泡的含量和外侧溶液是相同的，因此，抗生素卡纳霉素被添加到外部溶液中，以抑制蛋白质表达。作为一种控制，卡那霉素也添加到肿胀溶液中，在这种情况下，囊泡内的蛋白质表达被抑制。这表明卡那霉素不会通过膜扩散，并且一直抑制内部表达。然后进行F FRET测定，以证明ELP融入膜。在膜 ELP 的表达中，额外的肽融入膜中，这增加了 FRET 对之间的平均距离，从而导致供体信号的增加。提出的技术可用于产生简单的反应容器或制造具有肽基膜的人造细胞。可根据需要选择内部内容。通过使用这些肽囊泡，我们现在可以期待其中更复杂的合成反应。

Research

JoVE Journal

Bioengineering

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

轴突伸展生长：神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

科研

生物工程

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Generation and Recovery of ÃƒÅ½Ã‚Â²-cell Spheroids From Step-growth PEG-peptide Hydrogels

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

管式膜网络在心肌细胞的心房和心室分析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...