Scientists use deep mutational scanning to simultaneously test how multiple amino acid changes affect a protein of interest’s function. This technique relies on the generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure winnows the pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing is then used to measure the frequency of each variant during the selection process. Each variant is assigned a functional score based on its library frequency before selection compared to its library frequency after selection. Key to this process is the ability to generate full libraries of mutant proteins. Researchers from the Whitehead lab developed One pot saturation mutagenesis as a quick and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational scanning.
Writing a review article is a wonderful way to develop and exercise your scientist skill set. If you dread the thought of writing a review, or if you’re currently stuck trying to write one, hopefully this post will help you get things moving - remember you're becoming an expert in your field and are the perfect person to be writing the review! Doing so is a great way to develop your ability to write, to read efficiently, to search the literature, and to synthesize a large volume of information: basically, a scientist’s tool kit.
Epigenetic modifications are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation of the epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear. Today, targeted epigenetic modification at specific genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose!
Science rap mastermind, Tom McFadden, recently worked with high school students in the bay area to create a plasmid rap video for us (If you’re new to plasmids, we highly recommend checking out the video). Tom has made many more Science rap videos to teach students around the globe and is pushing SciComm further with his new company, Science with Tom. In this podcast, we learn more about Tom and pick his brain for advice on how to dive into new forms of science communication.
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).