Context-dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii.

Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5'atpA, and 3' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.

Red Fluorescent Protein Variant with a Dual-Peak Emission of Fluorescence.

The marine environment is a rich reservoir of diverse biological entities, many of which possess unique properties that are of immense value to biotechnological applications. One such example is the red fluorescent protein derived from the coral Discosoma sp. This protein, encoded by the DsRed gene, has been the subject of extensive research due to its potential applications in various fields. In the study, a variant of the red fluorescent protein was generated through random mutagenesis using the DsRed2 gene as a template. The process employed error-prone PCR (epPCR) to introduce random mutations, leading to the isolation of twelve gene variants. Among these, one variant stood out due to its unique spectral properties, exhibiting dual fluorescence emission at both 480 nm (green) and 550 nm (red). This novel variant was expressed in both Escherichia coli and zebrafish (Danio rerio) muscle, confirming the dual fluorescence emission in both model systems. One of the immediate applications of this novel protein variant is in ornamental aquaculture. The dual fluorescence can serve as a unique marker or trait, enhancing the aesthetic appeal of aquatic species in ornamental settings.

Reporter gene systems: A powerful tool for Leishmania studies.

Protozoan parasites of the genus Leishmania are responsible for leishmaniases, one of the most important anthropozoonotic diseases affecting millions of people worldwide. To date, there are no approved vaccines against leishmaniases for humans. At present, available treatment options lack specificity, which may lead to drug resistance and often cause adverse effects. Genomic analysis of Leishmania spp. revealed that most of the annotated genes encode hypothetical proteins, yet the functions of those proteins are still unknown. Characterization of these proteins is, hence, of utmost importance for the discovery of new therapeutic targets against leishmaniases. Reporter gene systems, or reporters, are powerful tools that enable the detection and measurement of targeted gene expression when introduced to a biological system. Over the years, numerous expression systems containing various reporters have been employed in characterizing several novel genes essential for parasite development. Such systems can be used to predict the subcellular localization of targeted proteins, screen antileishmanial drugs, and monitor the progression of infection within the vector and vertebrate hosts, among other uses. Therefore, it is critical to comprehend the available reporter gene expression systems to choose the most suitable for each study.