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Protocol for the transformation and engineering of edible algae Arthrospira platensis to generate heterologous protein-expressing strains.

ABSTRACT: Here, we present a protocol for harnessing the natural transformability of the edible algae Arthrospira platensis (common name: spirulina) to generate strains that express heterologous proteins. We describe the preparation of plasmids and the steps to grow A. platensis. We then detail the transformation and passage of the strains, followed by genomic DNA extraction and genotyping to assess integration of the gene of interest. This simple transformation protocol can be applied to genome manipulation of edible algae. For complete details on the use and execution of this protocol, please refer to Jester et al. (2022).¹.

SUBMITTER: Tabakh H

PROVIDER: S-EPMC9929480 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Protocol for the transformation and engineering of edible algae Arthrospira platensis to generate heterologous protein-expressing strains.

Tabakh Hannah H Jester Benjamin W BW Zhao Hui H Kuestner Rolf R Khuong Nhi N Shanitta Chelsea C Takeuchi Ryo R Roberts James J

STAR protocols 20230208 1

Here, we present a protocol for harnessing the natural transformability of the edible algae Arthrospira platensis (common name: spirulina) to generate strains that express heterologous proteins. We describe the preparation of plasmids and the steps to grow A. platensis. We then detail the transformation and passage of the strains, followed by genomic DNA extraction and genotyping to assess integration of the gene of interest. This simple transformation protocol can be applied to genome manipulat ...[more]

PMID: 36853691

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Project description:Phycocyanin and allophycocyanin are important active substances in Arthrospira platensis, because of their fluorescent characteristic and antioxidant capacity. In order to solve the problem of insufficient production and inconvenient modification of natural protein, recombinant expression was performed and the fluorescence activity and antioxidant activity was analyzed to meet the demand for phycocyanin and allophycocyanin. A total of seven recombinant strains were constructed in this study, including individual phycocyanin or allophycocyanin, co-expression of phycocyanin-allophycocyanin, and their co-expression with chromophore, and the expression strain for individual chromophore. Different molecular weights of phycocyanin and allophycocyanin were detected in the recombinant strains, which indicated the different polymers expressed. Through mass spectrometry identification, phycocyanin and allophycocyanin may form a dimer of 66 kDa and a polymer of 300 kDa. The results of fluorescence detection showed that phycocyanin and allophycocyanin combined with phycocyanobilin to show fluorescence activity. The fluorescence peak of recombinant phycocyanin was mainly concentrated at 640 nm, which was similar to natural phycocyanin, the fluorescence peak of purified recombinant allophycocyanin was at about 642 nm. The fluorescence peak of the co-expressed recombinant phycocyanin-allophycocyanin is located at 640 nm, and the fluorescence intensity is between the recombinant phycocyanin and the recombinant allophycocyanin. After purification, the fluorescence peak of the recombinant phycocyanin is more concentrated and the fluorescence intensity is higher, which is about 1.3 times of recombinant phycocyanin-allophycocyanin, 2.8 times of recombinant allophycocyanin, indicating that phycocyanin may be more suitable to be used as fluorescence probe in medicine. The antioxidant capacity was measured by using total antioxidant capacity (T-AOC) and DPPH (2,2'-diphenyl-1-triphenylhydrazino) free radical scavenging method, and the recombinant phycobiliprotein showed antioxidant activity. Phycocyanobilin also has certain antioxidant activity and could enhance the antioxidant activity of phycobiliprotein to a certain extent. Recombinant phycocyanin-allophycocyanin polymer has stronger T-AOC, which is about 1.17-2.25 times that of the other five recombinant proteins. And recombinant phycocyanin has stronger DPPH antioxidant activity, which is about 1.2-2.5 times that of the other five recombinant proteins. This study laid the foundation for the application of recombinant phycocyanin and allophycocyanin in medical detection and drug development.

Project description:Background Arthrospira platensis is farmed worldwide due to its nutrient-rich properties and provides multiple benefits to human health. However, the wide usage of silver nanoparticles (Ag NPs) causes pollution which may affect the nutritional quality of A. platensis. Hence, this study aimed to investigate the interaction and accumulation of Ag NPs on A. platensis, and determine the changes in biomass and nutritional value of A. platensis due to the exposure to Ag NPs. Methods The interaction and accumulation of Ag NPs on A. platensis were examined through Fourier transformed infrared (FTIR) spectroscopy and scanning electron microscope (SEM). The loss in biomass together with the macromolecules, pigments, and phenolic compounds of A. platensis was investigated upon treating with various concentrations of Ag NPs (5, 10, 25, 50 and 100 µg/mL) for 24, 48, 72 and 96 h. Results The results showed that the treatment of A. platensis with Ag NPs caused a dose and time-dependent reduction in biomass, macronutrients, pigments and phenolic compounds. The highest detrimental effects were found at 96 h with the reported values of 65.71 ± 2.79%, 67.21 ± 3.98%, 48.99 ± 4.39% and 59.62 ± 3.96% reduction in biomass, proteins, carbohydrates and lipids, respectively, along with 82.99 ± 7.81%, 67.55 ± 2.63%, 75.03 ± 1.55%, and 63.43 ± 2.89% loss in chlorophyll-a, carotenoids, C-phycocyanin, and total phenolic compounds of A. platensis for 100 µg/mL of Ag NPs. The EDX analysis confirmed the surface accumulation of Ag NPs on Arthrospira cells, while SEM images evidenced the surface alterations and damage of the treated cells. The functional groups such as hydroxyl, amine, methyl, amide I, amide II, carboxyl, carbonyl and phosphate groups from the cell wall of the A. platensis were identified to be possibly involved in the interaction of Ag NPs with A. platensis. Conclusion The study confirmed that the exposure of Ag NPs is detrimental to A. platensis where the interaction and accumulation of Ag NPs on A. platensis caused reduction in biomass, macromolecules, pigments, and total phenolic compounds.

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Protocol for the transformation and engineering of edible algae Arthrospira platensis to generate heterologous protein-expressing strains.

Publications

Protocol for the transformation and engineering of edible algae Arthrospira platensis to generate heterologous protein-expressing strains.

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