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Jin, H., Lindblad, P. & Bhaya, D. (2019). Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803. ACS Synthetic Biology, 8(4), 655-660
Open this publication in new window or tab >>Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803
2019 (English)In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 8, no 4, p. 655-660Article in journal (Refereed) Published
Abstract [en]

To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high, GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc10 and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
Keywords
Synechocystis sp. PCC6803, T7 RNA polymerase/T7 promoter circuit, orthogonal promoter, nickel inducible promoter
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-383202 (URN)10.1021/acssynbio.8b00515 (DOI)000465487100006 ()30935196 (PubMedID)
Available from: 2019-07-23 Created: 2019-07-23 Last updated: 2019-07-23Bibliographically approved
Lindblad, P., Fuente, D., Borbe, F., Cicchi, B., Conejero, J. A., Couto, N., . . . Wuenschiers, R. (2019). CyanoFactory, a European consortium to develop technologies needed to advance cyanobacteria as chassis for production of chemicals and fuels. Algal Research, 41, Article ID 101510.
Open this publication in new window or tab >>CyanoFactory, a European consortium to develop technologies needed to advance cyanobacteria as chassis for production of chemicals and fuels
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2019 (English)In: Algal Research, ISSN 2211-9264, Vol. 41, article id 101510Article, review/survey (Refereed) Published
Abstract [en]

CyanoFactory, Design, construction and demonstration of solar biofuel production using novel (photo) synthetic cell factories, was an R&D project developed in response to the European Commission FP7-ENERGY-2012-1 call "Future Emerging Technologies" and the need for significant advances in both new science and technologies to convert solar energy into a fuel. CyanoFactory was an example of "purpose driven" research and development with identified scientific goals and creation of new technologies. The present overview highlights significant outcomes of the project, three years after its successful completion. The scientific progress of CyanoFactory involved: (i) development of a ToolBox for cyanobacterial synthetic biology; (ii) construction of DataWarehouse/Bioinformatics web-based capacities and functions; (iii) improvement of chassis growth, functionality and robustness; (iv) introduction of custom designed genetic constructs into cyanobacteria, (v) improvement of photosynthetic efficiency towards hydrogen production; (vi) biosafety mechanisms; (vii) analyses of the designed cyanobacterial cells to identify bottlenecks with suggestions on further improvements; (viii) metabolic modelling of engineered cells; (ix) development of an efficient laboratory scale photobioreactor unit; and (x) the assembly and experimental performance assessment of a larger (1350 L) outdoor flat panel photobioreactor system during two seasons. CyanoFactory - Custom design and purpose construction of microbial cells for the production of desired products using synthetic biology - aimed to go beyond conventional paths to pursue innovative and high impact goals. CyanoFactory brought together ten leading European partners (universities, research organizations and enterprises) with a common goal - to develop the future technologies in Synthetic biology and Advanced photobioreactors.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Cyanobacterial synthetic biology toolbox, DataWarehouse, Chassis robustness, Biosafety, Improved electron chain, Large-scale photobioreactor cultivation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390421 (URN)10.1016/j.algal.2019.101510 (DOI)000472593800014 ()
Funder
EU, FP7, Seventh Framework Programme, 308518
Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-08-12Bibliographically approved
Eungrasamee, K., Miao, R., Incharoensakdi, A., Lindblad, P. & Jantaro, S. (2019). Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 6803. Biotechnology for Biofuels, 12, 1-13, Article ID 8.
Open this publication in new window or tab >>Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 6803
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2019 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 12, p. 1-13, article id 8Article in journal (Refereed) Published
Abstract [en]

Background

Cyanobacteria are potential sources for third generation biofuels. Their capacity for biofuel production has been widely improved using metabolically engineered strains. In this study, we employed metabolic engineering design with target genes involved in selected processes including the fatty acid synthesis (a cassette of accD, accA, accC and accB encoding acetyl-CoA carboxylase, ACC), phospholipid hydrolysis (lipA encoding lipase A), alkane synthesis (aar encoding acyl-ACP reductase, AAR), and recycling of free fatty acid (FFA) (aas encoding acyl-acyl carrier protein synthetase, AAS) in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Results

To enhance lipid production, engineered strains were successfully obtained including an aas-overexpressing strain (OXAas), an aas-overexpressing strain with aar knockout (OXAas/KOAar), and an accDACB-overexpressing strain with lipA knockout (OXAccDACB/KOLipA). All engineered strains grew slightly slower than wild-type (WT), as well as with reduced levels of intracellular pigment levels of chlorophyll a and carotenoids. A higher lipid content was noted in all the engineered strains compared to WT cells, especially in OXAas, with maximal content and production rate of 34.5% w/DCW and 41.4mg/L/day, respectively, during growth phase at day 4. The OXAccDACB/KOLipA strain, with an impediment of phospholipid hydrolysis to FFA, also showed a similarly high content of total lipid of about 32.5% w/DCW but a lower production rate of 31.5mg/L/day due to a reduced cell growth. The knockout interruptions generated, upon a downstream flow from intermediate fatty acyl-ACP, an induced unsaturated lipid production as observed in OXAas/KOAar and OXAccDACB/KOLipA strains with 5.4% and 3.1% w/DCW, respectively.

Conclusions

Among the three metabolically engineered Synechocystis strains, the OXAas with enhanced free fatty acid recycling had the highest efficiency to increase lipid production.

Keywords
Total lipid, Unsaturated lipid, Synechocystis sp, PCC 6803, Acyl-acyl carrier protein synthetase, Lipase A, Acyl-ACP reductase, Acetyl-CoA carboxylase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-374428 (URN)10.1186/s13068-018-1349-8 (DOI)000454948200008 ()30622650 (PubMedID)
Available from: 2019-01-28 Created: 2019-01-28 Last updated: 2019-01-28Bibliographically approved
Douchi, D., Liang, F., Cano, M., Xiong, W., Wang, B., Maness, P.-C., . . . Yu, J. (2019). Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria. Frontiers in Microbiology, 10, Article ID 1356.
Open this publication in new window or tab >>Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria
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2019 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1356Article in journal (Refereed) Published
Abstract [en]

Photosynthesis uses solar energy to drive inorganic carbon (Ci) uptake, fixation, and biomass formation. In cyanobacteria, Ci uptake is assisted by carbon concentrating mechanisms (CCM), and CO2 fixation is catalyzed by RubisCO in the Calvin-Benson-Bassham (CBB) cycle. Understanding the regulation that governs CCM and CBB cycle activities in natural and engineered strains requires methods and parameters that quantify these activities. Here, we used membrane-inlet mass spectrometry (MIMS) to simultaneously quantify Ci concentrating and fixation processes in the cyanobacterium Synechocystis 6803. By comparing cultures acclimated to ambient air conditions to cultures transitioning to high Ci conditions, we show that acclimation to high Ci involves a concurrent decline of Ci uptake and fixation parameters. By varying light input, we show that both CCM and CBB reactions become energy limited under low light conditions. A strain over-expressing the gene for the CBB cycle enzyme fructose-bisphosphate aldolase showed higher CCM and carbon fixation capabilities, suggesting a regulatory link between CBB metabolites and CCM capacity. While the engineering of an ethanol production pathway had no effect on CCM or carbon fixation parameters, additional fructose-bisphosphate aldolase gene over-expression enhanced both activities while simultaneously increasing ethanol productivity. These observations show that MIMS can be a useful tool to study the extracellular Ci flux and how CBB metabolites regulate Ci uptake and fixation.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2019
Keywords
MIMS, carbon uptake rate, cyanobacteria, FbaA, carbon fixation
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-390822 (URN)10.3389/fmicb.2019.01356 (DOI)000473067000001 ()31293533 (PubMedID)
Funder
Swedish Energy Agency, P46607-1
Available from: 2019-08-15 Created: 2019-08-15 Last updated: 2019-08-15Bibliographically approved
Towijit, U., Songruk, N., Lindblad, P., Incharoensakdi, A. & Jantaro, S. (2018). Co-overexpression of native phospholipid-biosynthetic genes plsX and plsC enhances lipid production in Synechocystis sp PCC 6803. Scientific Reports, 8, Article ID 13510.
Open this publication in new window or tab >>Co-overexpression of native phospholipid-biosynthetic genes plsX and plsC enhances lipid production in Synechocystis sp PCC 6803
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 13510Article in journal (Refereed) Published
Abstract [en]

The overexpression of native plsX and plsC genes involving in fatty acid/phospholipid synthesis first timely-reported the significantly enhanced lipid contents in Synechocystis sp. PCC 6803. Growth rate, intracellular pigment contents including chlorophyll a and carotenoids, and oxygen evolution rate of all overexpressing (OX) strains were normally similar as wild type. For fatty acid compositions, saturated fatty acid, in particular palmitic acid (16:0) was dominantly increased in OX strains whereas slight increases of unsaturated fatty acids were observed, specifically linoleic acid (18:2) and alpha-linolenic acid (18:3). The plsC/plsX-overexpressing (OX + XC) strain produced high lipid content of about 24.3% w/dcw under normal condition and was further enhanced up to 39.1% w/dcw by acetate induction. This OX + XC engineered strain was capable of decreasing phaA transcript level which related to poly-3-hydroxybutyrate (PHB) synthesis under acetate treatment. Moreover, the expression level of gene transcripts revealed that the plsX-and plsC/plsX-overexpression strains had also increased accA transcript amounts which involved in the irreversible carboxylation of acetyl-CoA to malonyl-CoA. Altogether, these overexpressing strains significantly augmented higher lipid contents when compared to wild type by partly overcoming the limitation of lipid production.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-365647 (URN)10.1038/s41598-018-31789-5 (DOI)000444086700022 ()30201972 (PubMedID)
Available from: 2018-11-14 Created: 2018-11-14 Last updated: 2018-11-14Bibliographically approved
Liang, F., Englund, E., Lindberg, P. & Lindblad, P. (2018). Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio. Metabolic engineering, 46, 51-59
Open this publication in new window or tab >>Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio
2018 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 46, p. 51-59Article in journal (Refereed) Published
Abstract [en]

The Calvin-Benson-Bassham (CBB) cycle is the main pathway to fix atmospheric CO2 and store energy in carbon bonds, forming the precursors of most primary and secondary metabolites necessary for life. Speeding up the CBB cycle theoretically has positive effects on the subsequent growth and/or the end metabolite(s) production. Four CBB cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), transketolase (TK) and aldolase (FBA) were selected to be co-overexpressed with the ethanol synthesis enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in the cyanobacterium Synechocystis PCC 6803. An inducible promoter, PnrsB, was used to drive PDC and ADH expression. When PnrsB was induced and cells were cultivated at 65 µmol photons m−2 s−1, the RuBisCO-, FBP/SBPase-, TK-, and FBA-expressing strains produced 55%, 67%, 37% and 69% more ethanol and 7.7%, 15.1%, 8.8% and 10.1% more total biomass (the sum of dry cell weight and ethanol), respectively, compared to the strain only expressing the ethanol biosynthesis pathway. The ethanol to total biomass ratio was also increased in CBB cycle enzymes overexpressing strains. This study experimentally demonstrates that using the cells with enhanced carbon fixation, when the product synthesis pathway is not the main bottleneck, can significantly increase the generation of a product (exemplified with ethanol), which acts as a carbon sink.

Keywords
Cyanobacteria, Biofuel, Carbon fixation, Ethanol, RuBisCO, FBP/SBPase, TK, FBA
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-351092 (URN)10.1016/j.ymben.2018.02.006 (DOI)000427934100007 ()29477858 (PubMedID)
Funder
Swedish Energy Agency
Available from: 2018-05-18 Created: 2018-05-18 Last updated: 2018-05-18Bibliographically approved
Liang, F., Lindberg, P. & Lindblad, P. (2018). Engineering photoautotrophic carbon fixation for enhanced growth and productivity. Sustainable Energy & Fuels, 2(12), 2583-2600
Open this publication in new window or tab >>Engineering photoautotrophic carbon fixation for enhanced growth and productivity
2018 (English)In: Sustainable Energy & Fuels, ISSN 2398-4902, Vol. 2, no 12, p. 2583-2600Article, review/survey (Refereed) Published
Abstract [en]

Oxygenic photosynthesis is the origin of most organic carbon compounds on Earth and an essential part of the natural carbon cycle. Cyanobacteria, the only oxygenic photoautotrophic prokaryotes, are important in several natural processes: as primary sustainable producers, in providing oxygen to the atmosphere, and in nitrogen fixation. From a biotechnological perspective, cyanobacteria are ideal cell factories since (i) the required energy and carbon source, sunlight and CO2, are abundant and freely available, (ii) cyanobacteria are capable of producing a variety of natural products, which can be used as fuels, medicines, cosmetics etc., and (iii) metabolic engineering and synthetic biology tools of cyanobacteria are being developed rapidly, making them feasible as host organisms for heterologous production of interesting compounds. However, compared to commercially employed heterotrophic microorganisms, the growth and productivity of cyanobacteria are currently not competitive. Therefore, improving cyanobacterial growth and productivity is an important task to enable commercialization of cyanobacterial bioproducts. Such studies also offer important clues for increasing the photosynthesis and yield of crop plants, which is important in view of providing food for a rapidly increasing world population. There are many strategies targeting this task, such as optimizing cultivation conditions, engineering native pathways, and introducing synthetic pathways based on an understanding of overall metabolic networks. One major limitation of cyanobacterial productivity, however, is the low efficiency of carbon fixation through the Calvin-Benson-Bassham (CBB) cycle. In this review, we introduce and discuss the possibilities to enhance growth and productivity by engineering the CBB cycle. We also give a brief discussion of options to further extend the capabilities of cells to fix inorganic carbon by the introduction of other native and artificial carbon fixation cycles.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2018
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-373527 (URN)10.1039/c8se00281a (DOI)000451078300003 ()
Funder
NordForsk
Available from: 2019-01-15 Created: 2019-01-15 Last updated: 2019-01-15Bibliographically approved
Maswanna, T., Phunpruch, S., Lindblad, P. & Maneeruttanarungroj, C. (2018). Enhanced hydrogen production by optimization of immobilized cells of the green alga Tetraspora sp CU2551 grown under anaerobic condition. Biomass and Bioenergy, 111, 88-95
Open this publication in new window or tab >>Enhanced hydrogen production by optimization of immobilized cells of the green alga Tetraspora sp CU2551 grown under anaerobic condition
2018 (English)In: Biomass and Bioenergy, ISSN 0961-9534, E-ISSN 1873-2909, Vol. 111, p. 88-95Article in journal (Refereed) Published
Abstract [en]

The green alga Tetraspora sp. CU2551 has previously been identified and characterized as a photosynthetic microorganism with high potential for H-2 production. In the present study, cells of Tetraspora CU2551 were entrapped and immobilized in an alginate matrix with the aim to analyze the effect of cell stacking and a reduced exposure of O-2 to the cells. The results showed that the most favorable immobilization conditions were 4% (w/v) of final alginate concentration and a cell concentration of 0.125 mg cell dry wt/mL alginate with a bead diameter of 2.80-3.35 mm. The H-2 production yields increased when the immobilized cells were incubated in S-deprived medium and this could be repeated at least for 3 production times. Maximal total H-2 production reached 7.68 +/- 0.88 mL H-2/25 mL medium, corresponding to a rate of 1182.45 +/- 24.40 nmol H-2/h/mg DW. This production is about 6 times higher compared to by cells in suspension, 2-10 times higher when compared to by other green algae, and 10-50 times higher when comparing with cyanobacteria. Based on our observations, immobilized cells of Tetraspora CU2551 is considered a very promising biological system for significant photobiological H-2 production by a photosynthetic microorganism.

Place, publisher, year, edition, pages
PERGAMON-ELSEVIER SCIENCE LTD, 2018
Keywords
Alginate, Immobilization, Biohydrogen production, Green algae, Tetraspora sp CU2551
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-352703 (URN)10.1016/j.biombioe.2018.01.005 (DOI)000426994100011 ()
Funder
NordForsk, 82845
Available from: 2018-06-08 Created: 2018-06-08 Last updated: 2018-06-08Bibliographically approved
Miao, R., Xie, H. & Lindblad, P. (2018). Enhancement of photosynthetic isobutanol production in engineered cells of Synechocystis PCC 6803. Biotechnology for Biofuels, 11, Article ID 267.
Open this publication in new window or tab >>Enhancement of photosynthetic isobutanol production in engineered cells of Synechocystis PCC 6803
2018 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, article id 267Article in journal (Refereed) Published
Abstract [en]

Background: Cyanobacteria, oxygenic photoautotrophic prokaryotes, can be engineered to produce various valuable chemicals from solar energy and CO2 in direct processes. The concept of photosynthetic production of isobutanol, a promising chemical and drop-in biofuel, has so far been demonstrated for Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942. In Synechocystis PCC 6803, a heterologous expression of alpha-ketoisovalerate decarboxylase (Kivd) from Lactococcus lactis resulted in an isobutanol and 3-methyl-1-butanol producing strain. Kivd was identified as a bottleneck in the metabolic pathway and its activity was further improved by reducing the size of its substrate-binding pocket with a single replacement of serine-286 to threonine (Kivd(S286T)). However, isobutanol production still remained low. Results: In the present study, we report on how cultivation conditions significantly affect the isobutanol production in Synechocystis PCC 6803. A HCl-titrated culture grown under medium light (50 mu mol photons m(-2) s(-1)) showed the highest isobutanol production with an in-flask titer of 194 mg l(-1) after 10 days and 435 mg l(-1) at day 40. This corresponds to a cumulative isobutanol production of 911 mg l(-1), with a maximal production rate of 43.6 mg l(-1) day(-1) observed between days 4 and 6. Additional metabolic bottlenecks in the isobutanol biosynthesis pathway were further addressed. The expression level of Kivd(S286T) was significantly affected when co-expressed with another gene downstream in a single operon and in a convergent oriented operon. Moreover, the expression of the ADH encoded by codon-optimized slr1192 and co-expression of IlvC and IlvD were identified as potential approaches to further enhance isobutanol production in Synechocystis PCC 6803. Conclusion: The present study demonstrates the importance of a suitable cultivation condition to enhance isobutanol production in Synechocystis PCC 6803. Chemostat should be used to further increase both the total titer as well as the rate of production. Furthermore, identified bottleneck, Kivd, should be expressed at the highest level to further enhance isobutanol production.

Place, publisher, year, edition, pages
BioMed Central, 2018
Keywords
Synechocystis, Cumulative titer, Cultivation condition, Metabolic bottleneck, Co-expression
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-359862 (URN)10.1186/s13068-018-1268-8 (DOI)000445749100004 ()30275907 (PubMedID)
Funder
EU, Horizon 2020, 640720NordForsk, 82845
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2018-10-18Bibliographically approved
Wegelius, A., Khanna, N., Esmieu, C., Barone, G. D., Pinto, F., Tamagnini, P., . . . Lindblad, P. (2018). Generation of a functional, semisynthetic [FeFe]-hydrogenase in a photosynthetic microorganism. Energy & Environmental Science, 11(11), 3163-3167
Open this publication in new window or tab >>Generation of a functional, semisynthetic [FeFe]-hydrogenase in a photosynthetic microorganism
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2018 (English)In: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 11, no 11, p. 3163-3167Article in journal (Refereed) Published
Abstract [en]

[FeFe]-Hydrogenases are hydrogen producing metalloenzymes with excellent catalytic capacities, highly relevant in the context of a future hydrogen economy. Here we demonstrate the synthetic activation of a heterologously expressed [FeFe]-hydrogenase in living cells of Synechocystis PCC 6803, a photoautotrophic microbial chassis with high potential for biotechnological energy applications. H-2-Evolution assays clearly show that the non-native, semi-synthetic enzyme links to the native metabolism in living cells.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-371404 (URN)10.1039/c8ee01975d (DOI)000449843300006 ()
Funder
EU, European Research Council, 714102Swedish Energy Agency, 11674-5Knut and Alice Wallenberg Foundation, 2011.0067Wenner-Gren FoundationsNordForsk, 82845
Note

Adam Wegelius and Namita Khanna contributed equally to this work.

Available from: 2018-12-20 Created: 2018-12-20 Last updated: 2019-01-07Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-7256-0275

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