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

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.

Development of spirulina for the manufacture and oral delivery of protein therapeutics.

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter-a major cause of infant mortality in the developing world-prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

Structural rearrangements near the chromophore influence the maturation speed and brightness of DsRed variants.

The red fluorescent protein DsRed has been extensively engineered for use as an in vivo research tool. In fast maturing DsRed variants, the chromophore maturation half-time is approximately 40 min, compared to approximately 12 h for wild-type DsRed. Further, DsRed has been converted from a tetramer into a monomer, a task that entailed mutating approximately 20% of the amino acids. These engineered variants of DsRed have proven extremely valuable for biomedical research, but the structural basis for the improved characteristics has not been thoroughly investigated. Here we present a 1.7 A crystal structure of the fast maturing tetrameric variant DsRed.T4. We also present a biochemical characterization and 1.6 A crystal structure of the monomeric variant DsRed.M1, also known as DsRed-Monomer. Analysis of the crystal structures suggests that rearrangements of Ser69 and Glu215 contribute to fast maturation, and that positioning of the Lys70 side chain modulates fluorescence quantum yield. Despite the 45 mutations in DsRed.M1 relative to wild-type DsRed, there is a root-mean-square deviation of only 0.3 A between the two structures. We propose that novel intramolecular interactions in DsRed.M1 partially compensate for the loss of intermolecular interactions found in the tetramer.

Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis.

The identification of Brucella can be a time-consuming and labor-intensive process that places personnel at risk for laboratory-acquired infection. Here, we describe a real-time PCR assay for confirmation of presumptive Brucella isolates. The assay was designed in a multiplex format that will allow the rapid identification of Brucella spp., B. abortus, and B. melitensis in a single test.