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

We gratefully acknowledge financial support through the DFG TRR 235 (Emergence of Life, project P15), the European Research Council (grant agreement no. 694410 AEDNA), and the TUM International Graduate School for Science and Engineering IGSSE (project no. 9.05). We thank E. Falgenhauer for her help with sample preparation. We thank A. Dupin and M. Schwarz-Schilling for their help with the TX-TL system and useful discussions. We thank N. B. Holland for useful discussions.

1. Expression of Elastin-like Polypeptides
<ol>
	<li>Day 1: Preparation of a starter culture and supplies for peptide expression
	<ol>
		<li>Prepare and autoclave expression culture flasks (4 x 2.5 L) and 3 L of LB medium. For 1 L of LB medium, add 25 g of LB powder to 1 L of ultrapure water.</li>
		<li>Prepare a starter culture with 100 mL of LB medium, 50 &#181;L of sterile-filtered (0.22 &#181;m filter) chloramphenicol solution (25 mg/mL in EtOH), and 50 &#181;L of sterile-filtered (0.22 &#181;m fil...

<ol>
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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. 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Film rehydration is a common procedure for the creation of small unilamellar vesicles. The main source of failure is the wrong handling of the materials used in the procedure.

Initially, the ELPs are produced by E. coli cells. The yield after ELP purification can vary significantly depending on how carefully the protocol is conducted during its crucial steps. These are the inverse temperature cycling (ITC) step and th...

The creation of synthetic living cellular systems is usually approached from two different directions. In the top-down method, the genome of a bacterium is reduced to its essential components, ultimately leading to a minimal cell. In the bottom-up approach, artificial cells are assembled de novo from molecular components or cellular subsystems, which need to be functionally integrated into a consistent cell-like system.
In the de novo approach, compartmentalization of the necessary biochemical components is usually achieved using membranes made from phospholipids or fatty acids1,2,3,4. This is because &#34;modern&#34; cell membranes mainly consist of phospholipids, while fatty acids are regarded plausible candidates of prebiotic membrane enclosures5,6. For the formation of new membranes or to facilitate membrane growth, amphiphilic building blocks must be provided from the exterior7 or ideally through production within a membranous compartment using the corresponding anabolic processes4,8.
While lipid synthesis is a relatively complex metabolic process, peptides can be produced quite readily using cell-free gene expression reactions9,10. Hence, peptide membranes formed by amphiphilic peptides represent an interesting alternative to lipid membranes as enclosures for artificial cell mimics that are able to grow11.
Amphiphilic elastin-like di-block copolymers (ELPs) are an attractive class of peptides, which can serve as the building block for such membranes12. The basic amino acid sequence motif of ELPs is (GaGVP)n, where &#8220;a&#8221; can be any amino acid except for proline and &#8220;n&#8221; is the number of motif repeats13,14,15,16,17. ELPs have been created with a hydrophobic block containing mainly phenylalanine for a and a hydrophilic block mainly composed of glutamic acid11. Depending on a and solution parameters, such as pH and salt concentration, ELPs exhibit a so-called inverse temperature transition at temperature Tt, where the peptides undergo a fully reversible phase transition from a hydrophilic to hydrophobic state. The synthesis of the peptides can be easily implemented inside vesicles using the &#8220;TX-TL&#8221; bacterial cell extract11,18,19,20,21, which provides all necessary components for coupled transcription and translation reactions.
The TX-TL system was encapsulated together, with the DNA template encoding the ELPs into ELP vesicles utilizing dehydration-rehydration from glass beads as a solid support. The formation of vesicles occurs through rehydration of the dried peptides from the bead surface11. Other methods22 for vesicle formation can be used, which potentially show lower polydispersity and larger vesicle sizes (e.g., electro-formation, emulsion phase transfer, or microfluidics-based methods). To test the viability of the encapsulation method, transcription of the fluorogenic aptamer dBroccoli23 can alternatively be used11, which is less complex than gene expression with the TX-TL system.
Due to the expression of the membrane building blocks in vesiculo and their subsequent incorporation into the membrane, the vesicles start to grow11. Membrane incorporation of the ELPs can be demonstrated through a FRET assay. To this end, the ELPs used for formation of the initial vesicle population are be conjugated with fluorescent dyes in equal shares constituting a FRET pair. Upon expression of non-labeled ELPs in vesiculo and their incorporation into the membrane, the labeled ELPs in the membrane are diluted and consequently the FRET signal decreases11. As a versatile and common method for conjugation, copper catalyzed azide-alkyne cycloaddition is used. With the use of a stabilizing ligand such as tris(benzyltriazolylmethyl)-amine, the reaction can be carried out in an aqueous solution at a physiological pH without the hydrolysis of reactants11, which is appropriate for conjugation reactions involving peptides.
The following protocol presents a detailed description of the preparation for growing ELP-based peptidosomes. The expression of the peptides and vesicle formation using the glass beads method are described. Furthermore, it is described how to implement transcription of the fluorogenic dBroccoli aptamer and the transcription-translation reaction for protein expression inside the ELP vesicles. Finally, provided is a procedure for the conjugation of ELPs with fluorophores, which can be used to prove vesicle growth through a FRET assay11.

<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

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Vesicle production 
Figure 1 shows transmission electron microscopy (TEM) images of vesicles prepared with different swelling solutions and the glass beads method (also see Vogele et al.11). For the sample in Figure 1A, only PBS was used as swelling solution to prove the formation of vesicles and to determine their size. When TX-TL was used as swelling solution (Figure 1B), the vesicles ...

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

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

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

Research

JoVE Journal

Bioengineering

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Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

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Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

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