An oral toxicity assessment of a mosquito larvicidal transgenic algae (Chlamydomonas reinhardtii) using adult Zebrafish and its embryos.

Mosquito-borne diseases pose a global health threat, with pathogens like Malaria, Dengue fever, and others transmitted by mosquitoes. Our study focuses on evaluating the toxicity of genetically engineered mosquito larvicidal algae (Chlamydomonas reinhardtii) to non-target organisms, specifically Zebrafish. We conducted a 90-day experiment, feeding Zebrafish different combinations of larvicidal algae and commercial fish feed. Statistical analysis revealed no significant differences in mortality, allergenicity, or moribundity among groups. Hematology, molecular analysis, and necropsy showed no physiological differences. Our findings indicate that the transgenic algae (TN72.cry11Ba) had no adverse effects on adult Zebrafish or their larvae. This study confirmed the safety of algae on non-target organisms, such as zebrafish.

Accelerating Chloroplast Engineering: A New System for Rapid Generation of Marker-Free Transplastomic Lines of Chlamydomonas reinhardtii.

'Marker-free' strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter.

Cloning and expression of an anti-cancerous cytokine: human IL-29 gene in Chlamydomonas reinhardtii.

Green algae, Chlamydomonas reinhardtii, with low cultivation cost, absence of endotoxins and insusceptibility to human pathogens is emerging as a potential system for the future production of recombinant proteins. The recent development of molecular tools enabling recombinant protein expression in algae chloroplast has provided new research and advance opportunities for developing low-cost therapeutic proteins. In the present study, algae chloroplast expression system was evaluated for the recombinant production of an anti-cancerous therapeutic protein, Interleukin 29 (IL29). The IL29 gene was cloned into algae chloroplast expression vector (pSRSapI). After the transformation, the positive clones were screened for homoplasmy and the presence of the IL29 gene by spot test and PCR analysis, respectively. The expressed SDS-PAGE and western blotting assay characterized IL-29. The algae expressed IL-29 was biologically active in an anti-proliferating bioassay using HepG2 cells. The results suggest that the Chlamydomonas reinhardtii expression system is convenient, low-cost, eco-friendly, and safe to express IL29.

Expression of a mosquito larvicidal gene in chloroplast and nuclear compartments of Chlamydomonas reinhardtii.

As a part of the search for environment-friendly biocontrol of mosquito-borne diseases, mosquito larvicidal potential of Bacillus thuringiensis subsp. jegathesan (Btj) Cry toxins is explored for toxins with increased toxicity. Safe delivery of the Cry toxins to mosquito larvae in aquatic habitats is a major concern. This is because in water bodies Bacillus thuringiensis (Bt) protein formulations degrade by sunlight, can sink down and get adsorbed by the silt. So, because of its short persistence the toxin requires repeated applications at the given site. Therefore, an upcoming approach is incorporating the Bt toxins in Chlamydomonas reinhardtii (C. reinhardtii) because it is a food of mosquito larvae in water and its molecular toolkit is well investigated for foreign gene expression. The present work aimed to compare the feasibility of C. reinhardtii chloroplast and nuclear compartments for stable expression of Cry11Ba toxin as this is the most toxic Btj protein to date, lethal to different mosquito species. With chloroplast expression of cry11Ba gene we were able to generate marker-free C. reinhardtii strain stably expressing Cry11Ba protein and demonstrating mortality against Aedes aegypti larvae. Moreover, for nuclear expression linking the cry11Ba gene to zeocin via foot and mouth disease virus (FMDV) 2A peptide resulted in the selection of transformants with increased cry11Ba mRNA expression levels by semi-quantitative reverse transcriptase PCR. Obtained results lay a foundation for the C. reinhardtii chloroplast expression system to be used for genetic engineering with Bt toxins which possess enhanced toxicity.