In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Summary

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.

Abstract

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.

Introduction

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 acids^1,2

Protocol

1. Expression of Elastin-like Polypeptides

1. Day 1: Preparation of a starter culture and supplies for peptide expression
   1. 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.
   2. Prepare a starter culture with 100 mL of LB medium, 50 µL of sterile-filtered (0.22 µm filter) chloramphenicol solution (25 mg/mL in EtOH), and 50 µL of sterile-filtered (0.22 µm filter) carbenicillin solution (100 mg/mL in 50% EtOH and 50% ultrapure water).
   3. Add a small streak from a pre-made bacterial stock containing ....

Results

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 al.......

Discussion

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.......

Materials

List of materials used in this article

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