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Investigation of artificial cells containing the Par system for bacterial plasmid segregation and inheritance mimicry – Nature Communications

Materials

1-Palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) was obtained from Avanti Polar Lipids (USA). N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihex-adecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (NBD-PE) was obtained from Thermal Fisher Scientific (USA). Cy3 maleimide (nonsulfonated) was purchased from APExBIO (USA). A Bradford protein assay kit, SDS‒PAGE gel configuration kit, 4×SDS‒PAGE sample loading buffer, protease inhibitor cocktail for purification of His-tagged proteins, green fluorescent DNA marker dye, streptavidin magnetic beads, Tris-borate-EDTA buffer (TBE) and Tween-20 were purchased from Beyotime (China). Protein markers (14.4–-97.4 kDa), 2×SYBR Green PCR Master Mix, a Na+K+-ATPase assay kit, TE buffer (pH 8.0), deoxyribonuclease I (DNase I), deoxyribonucleic acid sodium salt from salmon testes, and dithiothreitol (DTT) were obtained from Solarbio (China). A Ni-NTA 6FF prepacked chromatographic column and DNA marker (100–5000 bp) were purchased from Sangon Biotech (China). The plasmid pET28a containing the ParM gene, the plasmid pET28a containing the ParR gene, the plasmid pBR322 containing the parC gene, the forward primer (primer 1) and the reverse primer (primer 2) of the parC gene, and the forward primer (primer 1’) and the reverse primer (primer 2’) of the parC-eGFP gene were obtained from Sangon Biotech (China). Yeast extract fermentation agent, tryptone, and agar were purchased from AOBOX (China). Lysozyme, isopropyl β-D-thiogalactoside (IPTG), and imidazole were purchased from Biotopped (China). Sucrose, glucose, magnesium chloride (MgCl2), sodium chloride (NaCl), adenosine 5′-triphosphate disodium salt (ATP), 8-hydroxypyrene-1,3,6-trisulfonic acid, methylcellulose, trisodium salt (HPTS) and bovine serum albumin (BSA) were purchased from Sigma (China). Phosphate-buffered saline (PBS) and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Corning (USA). Chlorine e6 (Ce6) was purchased from MREDA (USA). Millipore Milli-Q water with a resistivity of 18.2 MΩ·cm was used in the experiments.

Instruments

A gel imaging instrument (Amersham Imager 600, GE) was used to detect the bands of the extracted ParM, ParR, and parC. A microplate reader (Molecular Devices, SpectraMax iD3, Germany) was used to determine the protein concentration and phosphate release. A circular dichroism instrument (Chirascan, Applied Photophysics, UK) was used to analyse the secondary structure of ParR and ParR-parC. All fluorescence images were obtained with a fluorescence microscope (Olympus IX73, Japan) and a confocal laser scanning microscope (Olympus FV 3000, Japan). A UV-Vis spectrometer (Agilent Technologies Cary 60) was used to measure the concentration of ATP. A fluorescence spectrometer (PerkinElmer Fluorescence spectrometer LS 55) was used to measure the fluorescence emission spectrum of the eGFP synthesized in vitro. An automated cell counter (Countess 3, Thermo Fisher Scientific) was used to measure the concentration of the streptavidin beads.

Expression and purification of ParM and ParR

The ParM gene was synthesized, cloned, and inserted into the expression vector pET28a (a plasmid encoding an N-terminal 6-histidine tag) to generate the ParM plasmid (Supplementary Data 1), which was subsequently transformed into E. coli BL21 (DE3) cells at a certain concentration (~10 ng). After the cells were grown to an OD600 of ~0.8, 1 mM IPTG was added at 15 °C overnight to induce the expression of ParM. The cell pellets were collected by centrifugation at 2500 × g for 5 min and resuspended in a mixture of 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 0.2 mg/ml lysozyme, protease inhibitor (100X), 0.02 mg/ml DNase I, and 1 mM MgCl2 at pH 8.0. After lysing, the cell lysate supernatant was collected via centrifugation at 15,000 × g for 30 min and loaded onto a Ni-NTA 6FF prepacked chromatographic column that was preequilibrated with 50 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole at pH 8.0. The ParM was purified by eluting with a series of buffer solutions containing different concentrations (50, 100, 200, 300, 400, and 500 mM) of imidazole. The ParM in different fractions was analysed via SDS‒PAGE. The relatively pure ParM fraction (200 mM imidazole) was further purified by a size-exclusion chromatographic column in 260 mM sucrose, 30 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT, and 100 mM KCl at pH 7.5. The obtained ParM was concentrated and stored at −80 °C. The ParR gene was synthesized, cloned, and inserted into the expression vector pET28a to obtain the ParR plasmid (Supplementary Data 1). ParR was extracted using a method similar to that used for ParM. The purities of ParM and ParR were greater than 99%, as determined by densitometric analysis with ImageJ. The N-terminal 6-histidine tag was not cleaved before use. The extracted proteins were stored at −80 °C.

Labeling of ParM with fluorescent dyes

Five additional amino acids (GSKCK) were added to the C-terminus of the ParM gene for fluorescent labeling of the Cy3 maleimide dye. Cy3 maleimide dye (16 μM) was mixed with ParM solution (96 μM) for 10 min at 25 °C, followed by the addition of 10 mM DTT to quench the reaction. The free Cy3 maleimide dye molecules were removed by centrifugation in an ultrafiltration tube (10 kDa) at 15,000 × g for 20 min in a mixture of 260 mM sucrose, 30 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT, and 100 mM KCl at pH 7.5 three times. The samples were stored at −80 °C.

Coverslip preparation

The coverslips were cleaned by ultrasonication in dichloromethane for 15 min, dried under a stream of nitrogen, and rinsed in Milli-Q water before being immersed in piranha solution (70:30, v/v, H2SO4:H2O2) for 5 min. The coverslips were subsequently washed with Milli-Q water and ethanol. The coverslips were stored in ethanol until use. The polymerization of ParM and subsequent parC-beads segregation in solution were performed using these treated coverslips.

Splitting of ParM filaments by laser irradiation

ParM filaments were precisely cut into two parts, both in solution and inside the GUVs, using high-intensity laser irradiation (561 nm, 0.7 mW, 5 s) through laser scanning at the central area (~1 μm in diameter) of the filaments several times until full splitting was achieved. The laser intensity was measured by an optical densitometer (CEL-NP2000).

Preparation of biotinylated parC

The biotinylated forwards primer (5′-TTCCATATGTTGTTACCCGCCAAACAAAACCCA-3′) and the reverse primer (5′-CGGGATCCAGTTTGATGTTTGTTGAACCGTCATC-3′) were used to amplify the biotinylated parC gene (Supplementary Data 1) via the classical PCR technique. Specifically, a 50 μL PCR mixture [2×SYBR Green PCR mix (25 μL), primer 1 (10 μM, 5 μL), primer 2 (10 μM, 5 μL), template DNA from the plasmid pBR322 parC (10 μL), and ddH2O (5 μL)] was subjected to thermal cycling (5, 10, 15, 20, 30, and 40 cycles) under the following thermal conditions: 95 °C for 10 min, [95 °C for 20 s, 60 °C for 30 s, and 72 °C for 60 s]. Biotinylated parC was obtained and stored at 4 °C (Supplementary Fig. 27a, b). The biotinylated eGFP-parC DNA was prepared using a method similar to that used for biotinylated parC.

Preparation of beads modified with parC

Streptavidin magnetic beads were concentrated on a magnetic separation rack for 1 min and then washed three times with TBS buffer solution (20 mM Tris-HCl, 0.137 M NaCl, pH 7.5) by the magnetic separation method. Afterward, the streptavidin magnetic beads were resuspended in 200 μL of biotinylated parC solution for 30 min. The free biotinylated parC was removed by magnetic separation in a mixture of 10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, and 0.05% Tween-20, pH 7.5, three times (Supplementary Fig. 28a, b). The sample was resuspended in 50 µL of buffer solution (260 mM sucrose, 30 mM Tris-HCl, 2 mM MgCl2, 1 mM DTT, 100 mM KCl, pH 7.5) and stored at 4 °C. The size of the parC-beads was 1352 ± 206 nm according to the DLS data (Supplementary Fig. 29). The number of plasmids on each bead was estimated using the following calculations. The concentration of streptavidin beads was (1.653 ± 0.006) × 106/mL using an automated cell counter. The concentration of biotinylated parC DNA was 466 ± 24 ng/μL, as determined via UV‒vis spectroscopy. The mixture of streptavidin beads and biotinylated parC DNA was incubated for 30 min at room temperature. After magnetic separation, the supernatant was removed and the parC DNA concentration was determined to be 419 ± 1 ng/μL. The parC-beads were washed again using the same protocol. The parC DNA concentration in the supernatant was 1.17 ± 0.29 ng/μL. After the third wash, the concentration of parC DNA in the supernatant was 0. Thus, the total amount of plasmid bound to the beads was calculated to be 72191.5 ng by subtracting the remaining plasmid DNA in the supernatants (419 ng/μL × 50 μL + 1.17 ng/μL × 50 μL) from the added plasmid DNA (466 ng/μL × 200 μL). The average amount of parC DNA was ~0.87 ng when 72191.5 ng was divided over 82,650. This amount of DNA single beads was sufficient to trigger eGFP expression using PURE systems.

ParM ATPase activity assay

The ATPase assay for ParM was performed in accordance with the kit protocol. Under strong acid conditions, the phosphates released from ATP hydrolyzed by ParM react with ammonium molybdate to produce phosphomolybdate yellows, which are reduced by stannous oxide (SnO) to phosphomolybdate blue. The intensity at 660 nm of the solution is proportional to the phosphate concentration. The ATPase activity of ParM was calculated using the following Eq. 1:

$${ATPase; activity; of; ParM}=frac{{C}_{{standard}}*{V}_{{total}},*,({A}_{{experiment}}-{A}_{{control}})}{T*,left({A}_{{standard}}-{A}_{{blank}}right) * ({C}_{{ParM}} * {V}_{{sample}})}$$

(1)

where Aexperiment, Acontrol, Astandard, and Ablank represent the absorbance at 660 nm of the experimental group, control group, standard sample, and blank sample, respectively, with a Cstandard of 0.5 μmol/mL, a Vtotal of 0.25 mL, a Vsample of 0.1 mL, and a T of 10 min.

Electrophoretic mobility shift assays

parC (275 ng) was mixed with ParR (0, 24, 48, 140, 240, 480, and 720 µM) in a buffer solution (20 mM HEPES, 150 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mg/ml BSA, 0.1 μg/μL sonicated salmon sperm DNA, 5% glycerol, pH 7.6). These samples were incubated at room temperature for 30 min and then loaded on a 1.5% magnesium-agarose gel. Electrophoresis was conducted at 150 V for 2 h using running buffer (45 mM Tris-HCl, 45 mM boric acid, and 5 mM magnesium acetate). The results were recorded using an Amersham Imager 600 (GE).

Circular dichroism (CD) measurements

ParR (3.31 μM) and a mixture of ParR (2.96 μM) and parC (251.56 ng/μL) were analysed using a circular dichroism instrument. The spectra were recorded with 1 nm steps and a dwell time of 2 s per step in a wavelength range of 180–300 nm.

Preparation of ParM-containing GUVs

Giant unilamellar vesicles (GUVs) were fabricated by a water-in-oil (w/o) emulsion-transfer method48. Specifically, 1 mg of POPC and 0.05 mg of NBD-PE were dissolved in 2 mL of mineral oil (lipid-mineral oil solution). The aqueous solution contained 260 mM sucrose, 30 mM Tris-HCl, 9.6 μM ParM, 1 mM ATP (if necessary), 2 mM MgCl2, 1 mM DTT, and 100 mM KCl, pH 7.5. Then, 30 µL of aqueous solution was mixed with 300 µL of lipid-mineral oil solution by vortexing for 30 s to generate a water-in-oil emulsion solution. The emulsion solution was gently added to 200 µL of isotonic glucose solution in a 1.5 mL centrifuge tube, followed by centrifugation (10,000 × g, 30 min) at 4 °C. The GUVs were collected at the bottom of the tube and used for subsequent experiments. ParMRC-containing GUVs were prepared through a similar method as that used for the ParM-containing GUVs. Since the amount of eGFP plasmid on each bead (~0.87 ng) is sufficient for expression in GUVs containing the PURE system, we encapsulated two parC-beads in each GUV as much as possible; as a result, the beads bound to the two ends of ParM filaments, avoiding the free unbonded beads in the GUV.

Ce6-mediated HPTS influx

GUVs (containing 260 mM sucrose) were mixed with 1 mM HPTS (containing 260 mM glucose) supplemented with Ce6 at 1.8, 3.6, 5.3, or 10.4 μM. These samples were treated by laser irradiation at 405 nm with light intensities of 0, 0.1, 0.3, 0.5, 0.7, and 0.9 mW for 5 s. Fluorescence microscopy images of the HPTS inside the GUV were recorded by confocal laser scanning microscopy (Olympus FV 3000, Japan) as a function of time.

Ce6-mediated GUV division

GUVs (containing 260 mM sucrose) were mixed with 400 mM glucose solution to induce the deformation of the GUVs. Once GUVs were prolonged due to the osmotic pressure, 10 μM Ce6 was added to achieve GUV division by laser irradiation at 405 nm for 5 s.

Gene expression of enhanced green fluorescence protein (eGFP)

A total of 20 μL of the PUREfrex 2.1 system containing 8 μL of solution I (amino acids, NTPs, tRNAs, enzyme substrates, etc.), 1 μL of solution II (proteins), 2 μL of solution III (20 µM ribosomes), 2 μL of cysteine (3 mM), 1 μL of GSH (80 mM), 1 μL of the plasmid encoding eGFP (20 ng/μL), and 5 μL of nuclease-free water was encapsulated in the GUV by the emulsion method. After the GUVs were incubated at 37 °C, the expression of eGFP was observed by fluorescence microscopy as a function of time.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.