Rosella is a pH-sensitive fluorescent biosensor that was recently deposited with Addgene by Dr. Mark Prescott. This system was developed for monitoring and analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced by a lack of nutrients and targets cytosol and organelles to the vacuole/lysosome for degradation and recycling. The key to Rosella’s autophagy-sensing abilities is that its fluorescence emission spectra changes when it goes from a more neutral pH compartment, like the cytosol, to the higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy and how Rosella improves upon them.
What is an Aptamer?
Nearly 30 years ago, two independent groups, led by Jack Szostak and Larry Gold, developed methods for selecting and amplifying RNA sequences that could bind very specifically to target molecules. Using a technique called systematic evolution of ligands by exponential enrichment (SELEX), some 1010 oligonucleotides could be screened for their affinity to a wide range of non-nucleotide targets. These RNA molecules, which could bind their targets with high specificity and affinity, were eventually called aptamers, from the Latin aptus, meaning “to fit”. SELEX could be used to classify DNA aptamers as well, and over the course of the next two decades, these nucleotide-based ligand binders would prove to be highly adaptable tools.
Quick Announcement from the Plasmids 101 Team: In preparation for the release of Addgene's Fluorescent Protein eBook - our next couple of plasmids 101 posts will gain a healthy, fluorescent glow. Stay tuned for more fluorescence-based Plasmid 101 posts in the coming weeks!
In biology as in life, more is often better. More transcription factor binding sites in a promoter lead to higher transcriptional activation. Multiple nuclear localization signals (NLS) increase protein import into the nucleus. In developing their SunTag technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent signals. Named for the "stellar explosion SUperNova," SunTag can help you turn up the brightness in your fluorescent imaging experiments.
Colorful CRISPR technologies are helping researchers visualize the genome and its organization within the nucleus, also called the 4D nucleome. Visualizing specific loci has historically been difficult, as techniques like fluorescent in situ hybridization (FISH) and chromosome capture suffer from low resolution and can’t be used in vivo. Some researchers have used fluorescently tagged DNA-binding proteins to label certain loci, but this approach is not scalable for every locus...unlike CRISPR. Early CRISPR labeling techniques allowed researchers to visualize nearly any single genomic locus, and recent advances have allowed scientists to track multiple genomic loci over time using all the colors of the CRISPRainbow.
High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences into plasmid DNA. In high throughput cloning techniques, although the specific sequences of the genetic elements may differ (e.g., a set of various mammalian promoters), the same cloning procedure can be used to incorporate each element into the final construct. This strategy can be used to build vectors with diverse functions, and thus, is used in many biological fields. In synthetic biology for example, high-throughput cloning can be used to combine the functions of different genetic elements to generate non-natural tools such as novel biological circuits or sensors. Given the expanding palette of fluorescent proteins and the availability of powerful imaging technologies, the combination of multiple fluorescent protein sequences to develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one such technique and has been used to produce complex fluorescent reporter constructs. These fluorescent reporters can be used to detect structure and protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek and Neveu, 2015).