Thylakoid Containing Artificial Cells for the Inhibition Investigation of

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Thylakoid containing artificial cells for the inhibition investigation of light-driven electron transfer during photosynthesis Wei Zong, Xunan Zhang, Chao Li, and Xiaojun Han ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00045 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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ACS Synthetic Biology

Thylakoid containing artificial cells for the inhibition investigation of light-driven electron transfer during photosynthesis Wei Zong, Xunan Zhang, Chao Li, and Xiaojun Han* Keywords: giant unilamellar vesicles, thylakoid, electron transfer, light reaction, artificial cells ABSTRACT The fabrication of artificial cells containing nature components is challenging. Herein we construct a thylakoid containing artificial cell (TA-cell) by forming multicompartmental structure inside giant unilamellar vesicles (GUVs) using osmotic stress. The thylakoids are selectively loaded inside each compartment to mimic “chloroplast”. The TA-cells are able to carry out photosynthesis upon light on. The TA-cells keep their 50% functionality of electron transfer for 12 days, which is twice of those of free thylakoids. Using TA-cells the inhibition of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) and heavy metal ions (Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+) on the electron transfer process in TA-cells is systematically investigated. Their half maximal inhibitory concentration (IC50) values are 36.23 ± 1.87 µM, 0.02 ± 0.01 µM, 0.42 ± 0.08 µM, 0.82 ± 0.12 µM, 1.97 ± 0.21 µM, and 4.08 ± 0.18 µM respectively. Hg2+ is the most toxic ion for photosynthesis process amount these five heavy metal ions. This biomimetic system can be expand to study other process during the photosynthesis. The TA-cells pave a way to fabricated more complicated nature component containing artificial cells. Cells constitute the basic structural and functional unit of all organisms.[1, 2] The reconstitution of simplified cell model is important to better understand the complicated biological processes in cells.[1, 3-5] Over the past few years, great efforts have been made to create the artificial cells.[6, 7] A number of artificial cells have been proposed for the potential applications in biotechnical and biomedical field.[8, 9] An advanced artificial model that linked the process of self-replication of DNA with the self-reproduction of the vesicle compartment was constructed to understand the origin of life.[10] Artificial cells containing catalase were implanted 1

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into mice suffering congenital deficiency in catalase to prevent the animals from the damaging effects of oxidants.[11] One of the most fancy task of artificial cells is to investigate the function of organelles in the cells. In nature, photosynthesis is the basis of life which can convert solar energy into chemical energy. Chloroplast is the core for photosynthesis. This organelle performs lots of the primary processes, such as light capture and electron transport within thylakoids. Thylakoid membranes comprise protein complexes in a dynamic lipid matrix, which enable the efficient conversion of light energy into chemical energy.[12] Cyanobacteria or thylakoids were entrapped within porous silica gel, which demonstrates that through the immobilization of photosynthetic membranes, photocatalytic reactors are capable of biomimicking photosynthetic processes.[13, 14] Light reaction is the critical process of photosynthesis, which involves a series of electron transfer to produce protons and oxygen. The proton gradient enables ATPase to produce ATP for carbon-fixation reaction. The artificial photosynthesis systems mainly focus on the final products. Few artificial photosynthesis systems addressed the influence of heavy metal ion on electron transfer process in the light reactions.[15, 16] Herein, we demonstrated the formation of thylakoid containing artificial cell (TA-cell) induced by osmotic stress. Using TA-cells the inhibition study of heavy metal ions on light-driven electron transfer was investigated. This novel artificial cell model presents an important step forward towards the development of synthetic living cells and provides a platform to mimic complex biochemical processes. RESULTS AND DISCUSSION The results are presented following the chronology of the experiments, with first the isolation of thylakoids from fresh spinach, then the subsequent loading of the inner vesicle of giant unilamellar vesicles (GUVs) with thylakoids by osmotic stress to form TA-cells, followed by the inhibition study of light-driven electron transfer in TA-cells. Isolation of thylakoids 2


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The thylakoids were extracted from fresh spinach tissue (Figure 1a). In order to confirm

the

thylakoid

was

extracted

successfully.

Sodium

dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments were carried out. Thylakoids membrane consists of 60-65 wt% proteins and 35-40 wt% lipids.[17, 18] The results from SDS-PAGE analysis showed the existence of individual units of protein complexes from thylakoid membranes (Figure 1b). From the gel image, we can recognize that the light harvesting complexes (LHCII), the reaction center subunits (D1, D2, CP43, and CP47 protein), photosystem II (PS II) complex subunits (33 kDa protein) and photosystem I (PS I) complex subunits (PsaA/B protein).[19] In order to further confirm the success of the thylakoids extraction process, fresh thylakoid extractive was measured using UV-Visible spectrometer (Figure 1c) and fluorescence spectrometer (Figure 1d). There are three obvious adsorption peaks at 440 nm, 470 nm, and 683 nm as shown in Figure 1c. The peaks at 440 nm, 683 nm are the adsorption peaks of Chl a, while the peak at 470 nm is the adsorption peak of Chl b and carotenoid.[20] Figure 1d is the fluorescence emission spectrum of isolated thylakoids, which showed the emission peak at 685 nm corresponding to PS II.[21] These results indicated that the thylakoids were successfully extracted from spinach.

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Figure 1. a) Schematic illustration of the extraction of thylakoids from spinach. b) SDS-PAGE of the purified thylakoids. The protein standards are indicated on the left side. c) UV-Visible and d) fluorescence emission spectra of free thylakoids. Preparation of TA-cells With the free thylakoids, we are able to fabricate artificial cells containing “chloroplasts”. As shown in Figure 2a, the inner vesicles containing thylakoids are regarded as artificial “chloroplasts”. By adjusting the osmotic pressure, the GUVs were induced to form vesicle in vesicle structures. The effect of hypertonic stress on GUVs composed of POPC, DMPC, and DPPC, examining in particular their shape deformation was described previously.[22] The DMPC GUVs containing 30 mol% Chol was found to form vesicle in vesicle structures in a controlled way. It is well known that a plant cell contain many chloroplasts. Therefore, vesicles in vesicle structure was appropriate to mimic the plant cell structure. First, thylakoids were extracted from fresh spinach by pulverization, filtration, and centrifugation. Second, GUVs were produced in B1 (0.4 M sucrose, 20 mM MES pH 6.5, 15 mM NaCl, 1% (w/w) K3[Fe(CN)6]) solution by electroformation method. Third, the thylakoid solution was added into the GUV solution aiming to cause the osmotic shock to form the TA-cells. Osmotic stress has been extensively used to induce the shape deformation of unilamellar vesicles.[23-25] The driving force behind the formation of the TA-cells was the difference in osmotic stress inside and outside of GUVs. To balance the high osmotic stress of the outside solution, water moves out the membrane of GUV, which induced the formation of some separate inner vesicles. This mechanism allows balancing the osmotic stress and prevents bursting of the vesicles. The thylakoid solution and GUV solution were mixed together at 25℃, the number of inner vesicle increased with the increase of mixing time as shown in Figure 2b. It is noted that some GUVs deformed to the vesicle in vesicle structure at 4 minutes after mixing (Figure 2bi), while some TA-cells only contain one vesicle at 7 minutes (Figure 2bii). After 10 min mixing, the high percentage of TA-cells including multiple inner vesicles were obtained (Figure 2biii), which are remarkably stable for 4


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one months at 4℃. In order to confirm the existence of thylakoids inside the inner vesicles, the red filter was used to take fluorescent images. The red dots in the inset of Figure 2biii are thylakoids, since the thylakoids emit red fluorescence[26]. These TA-cells possess the same adsorption peaks and emission peak with those from the free thylakoid (Figure S1), which also confirm the success of loading thylakoids in the artificial cells. Thus the “chloroplast” containing artificial cells were fabricated.

Figure 2. (a) Schematic illustration of the formation of TA-cells. (b) The images of deformation of GUVs mixed with thylakoid solution by osmotic stress for 4 min (i), 7 min (ii) and 10 min (iii), respectively. The lipid bilayer was labelled with 1 mol% NBD-PE (green fluorescence). The inset is the image of thylakoids (red color) containing GUV. The scale bars are 20 µm in b i, ii, iii, and 10 µm in the inset in Figure b-iii. Light-driven electron transfer in TA-cells Following the successful fabrication of TA-cells, their light-driven electron transfer behaviour was investigated. The light - induced electron transfer activity was measured using reducing power assay by calculating the number of electron transfer in the reduction from Fe3+(CN-)6 to Fe2+(CN-)6. The concentration of thylakoid inside TA-cells plays important role for photosynthesis reaction, which was optimized subsequently. Chl was chosen to determine the concentration of thylakoids. It is found 5



that the normalized amount of transferred electrons rapidly increases against the overall concentration of Chl from 0 to 2.6 mg mL-1, followed by slow increase afterwards (Figure 3a). Therefore the overall concentration of Chl with 2.6 mg mL-1 was fixed for TA-cells. The light irradiation time was investigated subsequently to obtain optimized reaction time of 120 min (Figure S2). The TA-cells remained the nature activity of chloroplast, which was confirmed by the light-switch property of TA-cells, as shown in Figure 3b. Without light, there is no electron transfer from 0 to 30 min; while the normalized amount of electron transfer increases from 30 - 90 min with light on. Further light off and on, the electron transfer activity stopped and started correspondingly. The light-switch property of TA-cells is similar to that of free thylakoids (Figure 3c). It should be mentioned that these TA-cells are robust when stored at 4℃ in dark. Equal volume samples were measured to obtain the normalized amount of transferred electrons every day. TA-cells retained at least 50% photosynthesis activity for 12 days (Figure S3 and Table S1). The compartmentalized thylakoid in the TA-cell showed the crowded state after two weeks (Figure S4). On the contrary, the free thylakoids can retain the 50% photosynthesis activity only for 6 days. The sealed membrane structure prevents the damages of thylakoids, which is one of the advantages of artificial cells. 600

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Figure 3. Light-driven electron transfer plots. (a) The influence of overall Chl concentration on the normalized amount of electron transfer in free thylakoids. (b) TA-cells and (c) free thylakoids light switch properties. The inhibition of light driven electron transfer of light reaction

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Membrane proteins, especially ion channels are vital components of biomembranes for signalling, mass transport, and immune recognition.[27] Therefore, to demonstrate the feasibility of molecule transfer between subcompartments, a membrane protein melittin was inserted into the lipid bilayers to create nanopores (Figure S5a). Melittin self-assembles into bilayers to create pores of 1-3 nm in diameter depending on the number of assembly subunits, which allows small molecules to transfer.[28] With melittin in the bilayers, the rhodamine molecules were released quickly from inner vesicle to reach the final stage for 20 mins (Figure S5b). Meanwhile, without mellitin pores in the lipid bilayer, almost all rhodamine molecules were retained inside the vesicle (Figure S5c). With the mellitin pores in the lipid bilayers, DCMU and heavy metal ions were able to move inside the TA-cells to interact with thylakoids as shown in Figure 4a. DCMU is a kind of herbicide which inhibited the electron transfer in PSII. The mechanism is the site of QB was occupied by DCMU to prevent the electron transfer from QA (primary quinones of PSII) to QB (secondary quinones of PSII)[29, 30] as shown in Figure S6. The TA-cells were incubated with DCMU before light irradiation for 60 mins to allow the DCMU molecule going through the melitiin nanopore to interact with thylakoids. As expected, with the increase of DCMU concentration, the normalized amount of electron transfer gradually decreased. No electron transfer was detected at 100 µM DCMU as shown in Figure 4b. The software IBM-SPSS 13.0 was used to calculate the IC50 value. The IC50 of DCMU to TA-cells was 36.23 ± 1.87 µM, which was larger than that to free thylakoids (IC50=27.18 ± 1.49 µM). These results indicated that the TA-cells were suitable for the study of inhibition behavior of electron transfer of photosynthesis reaction; on the other hand, these results also indicated that the artificial chloroplast containing cell were able to protect the internal active components from damaging. The TA-cells are also a good platform to study the inhibition effect of heavy metal ions on photosynthesis reaction. Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+ were typical heavy metal pollutant ions to the plants. They disrupt many physiological functions by binding to protein sulfhydryl groups and substituting essential ions.[31] The heavy metal ions of the above mentioned were incubated with TA-cells for 5 hours to allow 7



these ions to penetrate the membranes through melittin nanopores. The normalized amount of transferred electrons were measured as a function of the concentration of each heavy metal ion, as shown in Figure 4c. Their IC50 values were calculated to be 0.02 ± 0.01 mM, 0.42 ± 0.08 mM, 0.82 ± 0.12 mM, 1.97 ± 0.21 mM, and 4.08 ± 0.18 mM for Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+, respectively (Figure 4d). Therefore the toxicity sequence on TA-cells from high to low is Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+. Those metal ions inhibit the electron transfer during the light reaction by denature the proteins.[32-34] In addition, the redox potential of Hg2+/Hg (0.80 V) is higher than Fe(CN)63-/Fe(CN)64- (0.36 V), which indicates that Hg2+ may also catch the electrons in the series of reaction during the photosynthesis apart from denaturing proteins.

Figure 4. (a) The schematic illustration of DCMU and heavy metal ions penetrating membranes through melittin nanopores to interact with thylakoids. (b) The concentration dependence of DCMU on the inhibition of light-induced electron transfer process of TA-cells. (c) The inhibition plots of heavy metal ions of Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+ on TA-cells. (d) The IC50 values of Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+. 8


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CONCLUSIONS We fabricated TA-cells using osmotic stress via the deformation of GUVs. This novel artificial cell model contains thylakoid containing compartments. Each thylakoid compartment can be regarded as an artificial chloroplast. The light-driven electron transfer conversion process was imitated using TA-cells. The inhibition from DCMU and heavy metal ions Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+ on the electron transfer in TA-cells was systematically investigated, which proves the TA-cells are potential platforms for detailed study of photosynthesis mechanism. More diversified nature component containing artificial cells can be fabricated using similar way to study complicated biological mechanisms in cells. METHODS

Materials and Characterization: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (Chol) were purchased from Avanti Polar Lipids (USA). Fluorescence-labeled 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) were obtained from Molecular Probes (Eugene, Oregon, US). Triton X-100, melittin (⩾97%), Albumin from bovine serum (BSA), Mercury dichloride (HgCl2), Cupric chloride (CuCl2), Cadmium chloride (CdCl2), Lead chloride (PbCl2) and Zinc chloride (ZnCl2) were obtained from Sigma (Beijing, China). 4-Morpholineethanesulfonic acid (MES), trihydroxymethyl methylglycine (Tricine) and 3-(3V,4V-dichlorophenyl)-1,1-dimethylurea) (DCMU) were purchased from Aladdin (Shanghai, China). The fresh spinach was purchased from local market. Sodium chloride (NaCl), sucrose, Iron (III) chloride hexahydrate (FeCl3·6H2O), Potassium ferricyanide (K3Fe(CN)6), Prussian blue (Fe4[Fe(CN)6]3), Disodium Hydrogen Phosphate Dodecahydrate (Na2HPO4·12H2O), Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) and Absolute ethyl ethanol were purchased from XiLong Chemical Co., Ltd (China). Trichloroacetic acid (TCA) was purchased from Tianjin Tianli Chemical Reagents Ltd (China). Biowest Agarose was purchased from Gene Company (Spain). 9



Glass slides coated with indium tin oxide (ITO, sheet resistance ≈ 8 to 12 Ω, thickness ≈ 160 nm) were purchased from Hangzhou Yuhong technology Co. Ltd (China). Millipore Milli-Q water with a resistivity of 18.0 MΩ cm was used for solution preparation. Fresh spinach leaves were smashed using a blender (Shanghai, China). Intact spinach cells were broken using Ultrasonic Homogenizer (JY92-IIN, Ningbo, China). The separation and purification of thylakoid were completed by centrifugation (TG16-WS, China). The membrane proteins on thylakoid were analyzed using western blotting system (Bio-Rad, USA). Cary 60 UV-Vis spectrophotometer (Agilent, USA) and fluorescence spectrometer (Perkin Elmer LS55) were used for UV-Vis spectra and fluorescence emission spectra. Microscopy images were taken with a fluorescence microscope (Nikon 80i, Japan). An XPA-VII type photochemical reactor (Xujiang Machine Factory, Nanjing, China) was used to provide the artificial sunlight.

Extraction of thylakoids from spinach: Thylakoids were obtained from fresh spinach. Spinach leaves were washed and weeded out veins. The thylakoid extraction protocol was described elsewhere,[35, 36] and depicted in Figure S7. Spinach leaves mixed with buffer solution B2 (0.4 M sucrose, 20 mM Tricine pH 7.8, 40 mM NaCl, 0.2% (w/w) BSA) and then homogenized in a precooled blender. Using multilayer precooled gauze to filter the liquid and collect the filtrate. The filtrate was centrifuged by centrifugation for 15 min with 6000 rpm at 4℃ to collect intact and broken chloroplasts. The supernatant was removed. The sediment was suspended in buffer solution B3 (20 mM Tricine pH 7.8, 10 mM NaCl, 0.2% (w/w) BSA) and dealt for 20 min with Ultrasonic Homogenizer to break the intact chloroplast. Following centrifugation at 10000 rpm for 15 min at 4℃, the sediment was suspended in buffer solution B4 (0.4 M sucrose, 20 mM MES pH 6.5, 15 mM NaCl). The chlorophyll (Chl) overall concentration of thylakoid membrane suspension was determined in 80% (v/v) acetone solutions as described by Arnon[37] and then adjusted using buffer B4. The obtained thylakoid solution was stored at 4℃ before use. 10


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Preparation of TA-cells: The GUV was prepared using electroformation method[38-43] as mentioned in Supplementary Section 7. Lipid solution (5mg mL-1) was composed of Chol, DMPC and NBD-PE at a 30:69:1 molar mass ratio. Melittin was dissolved in ethanol and further mixed with lipid solution with the final concentration of 1 µM. The lipid and melittin mixture solution was deposited on ITO electrode surface, followed by drying under vacuum for 2 h to remove the organic solvent. GUVs were produced after applying AC electric field for 4 hours in B1 solution. Then the thylakoid solution and GUVs solution (2/5, v/v) were mixed together for 10 min at 25 ℃ to obtain TA-cell by osmotic stress.

Light reaction in TA-cells: The light induced electron transfer activity was measured using reducing power assay by calculating direct electron transfer in the reduction of Fe(CN)63- to Fe(CN)64- as described previously[44] with some modifications. The Fe(CN)63- is able to catch all the electrons from the light induced electron transfer chain in the light reaction in thylakoid to form Fe(CN)64-, due to the high redox potential of Fe(CN)63-/Fe(CN)64- (0.36 V) (Figure S8). The generated amount of Fe(CN)64- is proportional to the whole amount of transferred electrons in the light reaction of certain amount of thylakoids, since enough Fe(CN)63- was added into the reaction solution. The amount of Fe(CN)64- was measured by forming Prussian blue complex (Fe4[Fe(CN)6]3), which is quantitated by measuring the absorbance intensity at λ680 nm (Figure S9a) using UV-Vis spectrophotometer. The relationship of Fe(CN)63-, Fe(CN)64- and Fe4[Fe(CN)6]3 is given below:

3 [Fe(CN)6]3-

+3e-

3+ 3 [Fe(CN)6]4- + 4 Fe Fe4[Fe(CN)6]3

From above relation, one Fe4[Fe(CN)6]3 existence means three electrons were transferred. Thus by measuring the amount of Fe4[Fe(CN)6]3, we are able to calculate the amount of transferred electrons during the light reaction. From the calibration curve of Fe4[Fe(CN)6]3 (Figure S9b), Cp was obtained. The normalized amount of transferred electrons can be calculated as below: Normalized amount of transferred electrons (n mol · (mg Chl)-1 · h-1) 11



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（1） where Cp (nM) is the concentration of Fe4[Fe(CN)6]3 obtained from Figure S9b, VP (L) is the corresponding Fe4[Fe(CN)6]3 volume, mchl (mg) means the mass of chl in those thylakoids where 3CP (nM)×Vp (L) electrons transferred during the photosynthesis , t (h) is the irradiation time. The real experimental procedure is described as below. As a control, the free thylakoid extracts (4 mL) and B1 solution (10 mL) were mixed together. The artificial photosynthesis was conducted with a 250 W xenon lamp at 20 ℃. The overall concentration of Chl (0.026 mg mL-1, 0.104 mg mL-1, 0.52 mg mL-1, 1.04 mg mL-1, 2.6 mg mL-1, 3.64 mg mL-1, 5.2 mg mL-1) and light irradiation time (30 min, 60 min, 90 min, 120 min, 150 min and 180 min) were systematically investigated. At the end of the reaction, 0.7 mL 2% (w/w) Triton X-100 was added into above mixture solution at 25 ℃ for 10 min to break the thylakoid membrane and release the all content inside thylakoid. 10 mL of 10% (w/v) TCA solution was added to the mixture solution at 25 ℃ for 5 min to digest the redundant K3Fe(CN)6 for eliminating the absorption interference from K3Fe(CN)6 yellow solution, followed by centrifuging at 3000 rpm for 10 min. 1.5 mL supernatant from above mixture solution was mixed with 1.5 mL buffer solution B4 and 0.1 mL of 0.1% (w/v) FeCl3 solution in a test tube at 25 ℃. 10 min later, the absorbance of above solution was measured at 680 nm to obtain the concentration of Fe4[Fe(CN)6]3 (Cp, nM). Before the artificial photosynthesis was conducted in TA-cells, the free thylakoids outside the TA-cells were removed through 10 µm filters. The fluorescence images before and after the TA-cells were filtered were shown in Figure S10. The TA-cell solution (14 mL, Chl 2.6 mg mL-1) was illuminated using a 250 W xenon lamp at 20 ℃ for 120 min. After light irradiation, the TA-cells were disrupted using 0.7 mL 2% (w/w) Triton X-100 at 25 ℃ for 10 min and 10 mL 10% (w/v). TCA solution was added into abovementioned solution, followed by centrifugation with a centrifuge at 3000 rpm for 10 min. 1.5 mL supernatant upper layer was mixed with equivoluminal 12


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0.1% (w/v) FeCl3 solution for 10 min at 25 ℃, and the concentration of Prussian blue was obtained by UV-vis spectrometer.

Inhibition of light-driven electron transfer in TA-cells：：DCMU is an inhibitor of electron transfer. The melittin containing TA-cell solution was mixed with DCMU solution (5/1, v/v) in darkness for 1 h at 25 ℃. The melittin molecules form pores in the membrane to allow DCMU/heavy metal ions to reach thylakoids inside the artificial chloroplasts. The final concentration of DCMU (10 µM, 30 µM, 50 µM and 100 µM) were used to study DCUM inhibition efficiency on the electron transfer in TA-cells. Heavy metal ions Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+ were the toxic ions in water. The intact melittin containing TA-cell solution was mixed with Hg2+, Cu2+, Cd2+, Pb2+ and Zn2+ solution (5/1, v/v) respectively in darkness for 5 h at 25 ℃. The final concentration of Hg2+ (0.1 mM, 0.2 mM and 0.3 mM), Cu2+ (0.2 mM, 0.4mM and 0.6 mM), Cd2+ (0.4 mM, 0.8 mM and 1.2 mM), Pb2+ (1 mM, 2 mM and 3 mM) and Zn2+ (2 mM, 4 mM and 6 mM) were used to obtain their half maximal inhibitory concentration (IC50) values. After co-culture, the abovementioned TA-cell solution was washed using B1 solution to remove the redundant DCMU/heavy metal ions by filtering them through 200 nm filter membrane for 5 times. The overall concentration of Chl in TA-cells was adjusted using B1 solution. The treated TA-cells were exposed under artificial light for 120 minutes. SUPPORTING INFORMATION The supporting Word document contains the supplementary material referenced in this text. AUTHOR PRESENT ADDRESS State Key Laboratory of Urban Water Resource and Environment, School of Chemistry and Chemical Engineering. Harbin Institute of Technology, 92 West Da-Zhi Street, Harbin, 150001, China E-mail: [email protected] CONFLICTS OF INTEREST The authors declare no competing financial interest. 13



AUTHOR CONTRIBUTIONS X.J.H. and W.Z. conceived of the project, designed all experiments, W.Z. and X.N.Z. performed all experiments, W.Z., X.N.Z. and C. L. performed all data analysis. X.J.H. wrote the paper, and all authors commented on the paper. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No.21773050, 21528501), and the Harbin Distinguished Young Scholars Fund (No. 2017RAYXJ024). REFERENCES [1] [2] [3] [4]

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Thylakoid Containing Artificial Cells for the Inhibition Investigation of

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