Scientific inquiry is a process that is revered as much as it is misunderstood. As I listed to a TED talk about the subject, I was reminded that for the general public the foundation of science is the scientific method—the linear process of making an observation, asking a question, forming an hypothesis, making a prediction and testing the hypothesis.
While this process is integral to doing science, what gives scientific findings credibility and value is consensus from the scientific community. Building consensus is the time-consuming process that includes peer review, publication and replication of results. It is also the part of scientific inquiry that so often leads the public to misunderstand and mistrust scientific findings.
Ribonucleoprotein complex with Cas9, guide RNA and donor ssDNA. Copyright Promega Corporation.
With the advent of genome editing using CRISPR-Cas9, researchers have been excited by the possibilities of precisely placed edits in cellular DNA. Any double-stranded break in DNA like that induced by CRISPR-Cas9 is repaired by one of two pathways: Non-homologous end joining (NHEJ) or homology-directed repair (HDR). Using the NHEJ pathway results in short insertions or deletions (indels) at the break site, so the HDR pathway is preferred. However, the low efficiency of HDR recombination to insert exogenous sequences into the genome hampers its use. There have been many attempts at boosting HDR frequency, but the methods compromise cell growth and behave differently when used with various cell types and gene targets. The strategy employed by the authors of an article in Communications Biology tethered the DNA donor template to Cas9 complexed with the ribonucleoprotein and guide RNA, increasing the local concentration of the donor template at the break site and enhancing homology-directed repair. Continue reading →
By US Environmental Protection Agency [Public domain], via Wikimedia Commons
February 11 is the International Day of Women and Girls in Science, a reminder that there is still a gender gap in science. Despite the obstacles that women need to overcome, their contributions to field of science have benefited not only their fellow researchers but also their fellow humans. From treatments for diseases to new discoveries that opened up entire fields, women have advanced knowledge across the spectrum of science. Below is a sampling of the achievements of just a few women. What other living female scientist or inventor might you add?
Hate malaria? You can thank Tu Youyou for discovering artemisinin and dihydroartemisinin, compounds that are used to treat the tropical disease and save numerous lives. Her discovery was so significant, she received the 2015 Nobel Prize in Physiology or Medicine. Continue reading →
Researchers having been sharing plasmids ever since there were plasmids to share. Back when I was in the lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself or you could “clone by phone”. One of my professors was excellent at phone cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted. Addgene makes this so much easier to share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research and discovery by improving access to useful research materials and information. Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are present in the repository. We encourage people to go to Addgene to get new innovative tools. Afterall, isn’t science better when we share?
I’d like to focus on some tools in the Addgene collection based on NanoLuc® Luciferase (NLuc). The creation of NanoLuc® Luciferase and the optimal substrate furimazine is a good story (1). From a deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase. This is important not so much for how bright you can make a reaction but for how sensitive you can make a reaction. NLuc requires 100-fold less protein to produce the same amount of light from a Fluc or RLuc reaction. NLuc lets you work at physiological concentrations. NLuc is bright enough to detect endogenous tagged genes generated through the CRISPR/Cas9 knock-in. NLuc is very inviting for endogenous tagging as it is only 19kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk, J.L. et al (2).
Two applications of NanoLuc® Technology have caught my attention through coupling the luciferase with fluorescent proteins to make better imaging reporters and biosensors. Continue reading →
This year’s participants in Emerging Techniques in Protein and Genetic Engineering, a two-credit graduate course offered in partnership with the Department of Oncology, UW-Madison, held July 17-21, 2017.
Today’s author extends thanks to Heather Gerard, Intellectual Property Manager, Promega Corporation for contributing her expertise to this post.
Students most often come to the BTC Institute with the primary goal of learning about molecular biology technologies. Our mission is to help them update their experimental tool-box, facilitating more capable studies of DNA, RNA and proteins back in their home laboratories.
There are new developments in genetics coming to light every day, each with the potential to dramatically change life as we know it. The increasingly controversial gene editing system, dubbed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), is at the root of it all. Harnessed for use in genome editing in 20131, CRISPR has given hope to researchers looking to solve various biological problems. It’s with this technology that researchers anticipate eventually having the means to genetically modify humans and rid society of genetic disorders, such as hemophilia. While this is not yet possible, the building blocks are steadily being developed. Most recently, two groundbreaking studies concerning CRISPR have been released to the public. Continue reading →
Recently a FaceBook friend of mine (who is not a scientist) shared a video from WIRED Science where the concept of CRISPR is explained at 5 Levels of Difficulty— for a 7 year old, a teenager, a college student, a grad student and a CRISPR expert.
First it was pretty amazing to me that my non-scientist friends are interested enough in learning about CRISPR to share this type of information—perhaps showing just how popular and exciting the method has become. People outside the scientific field are hearing a lot about it, and are curious to know more.
This video does a great job of explaining the technique for all its intended audiences. It also is a nice illustration of how to share information in an easily understandable format. Even with the 7 year old and 14 year old, the information is shared in a conversational way, with everyone involved contributing to sharing information about CRISPR.
CRISPR is a hot topic right now, and rightly so—it is revolutionizing research that relies on editing genes. But what exactly is CRISPR? How does it work? Why is everyone so interested in using it? Today’s blog is a beginner’s guide on how CRISPR works with an overview of some new applications of this technology for those familiar with CRISPR.
Introduction to CRISPR/Cas9
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were discovered in 1987, but it took 30 years before scientists identified their function. CRISPRs are a special kind of repeating DNA sequence that bacteria have as part of their “immune” system against invading nucleic acids from viruses and other bacteria. Over time, the genetic material from these invaders can be incorporated into the bacterial genome as a CRISPR and used to target specific sequences found in foreign genomes.
CRISPRs are part of a system within a bacterium that requires a nuclease (e.g. Cas9), a single guide RNA (sgRNA) and a tracrRNA. The tracrRNA recruits Cas9, while sgRNA binds to Cas9 and guides it to the corresponding DNA sequence of the invading genome. Cas9 then cuts the DNA, creating a double-stranded break that disables its function. Bacteria use a Protospacer Adjacent Motif, or PAM, sequence near the target sequence to distinguish between self and non-self and protect their own DNA.
While this system is an effective method of protection for bacteria, CRISPR/Cas9 has been manipulated in order to perform gene editing in a lab (click here for a video about CRISPR). First, the tracrRNA and sgRNA are combined into a single molecule. Then the sequence of the guide portion of this RNA is changed to match the target sequence. Using this engineered sgRNA along with Cas9 will result in a double-stranded break (DSB) in the target DNA sequence, provided the target sequence is adjacent to a compatible PAM sequence. Continue reading →
Summer on the Prairie at Promega–Study science surrounded by birds, bees, flowers and amazing prairie.
Summer, a much-looked forward to season. We typically pack in the activities and make the most of the daylight. We work hard and we play hard. This summer will be no exception, and at the BTC Institute, we are already getting set to host as many students as we can. We will see middle and high schoolers, K-12 teachers, college students, graduate students, college and university faculty and staff, and professionals in the biotech community under our roof at some point. You may want to join us too!
Our programs for advanced learners, geared toward the graduate student or biotech professional, offer much more than just a rigorous immersion in molecular biology theory and practice. Held at the BTC Institute at Promega Headquarters, they are taught by highly knowledgeable scientists, coming from both industry and academia. These instructors offer a wealth of information and share their expertise as well as life experiences with students. Informal discussions about career trajectories and access to industry are important added benefits to attending these off-campus workshops. Continue reading →
Microbial cells outnumber the cells of our own bodies approximately 10:1, these microbes that live on our skin and along the epithelial linings of our internal tubes make up our microbiota*, and they can have major effects on our health. Most of our microbiota are commensal organisms, living in harmony with our body, but if you suppress our immune system or greatly reduce their populations with large doses of antibiotics, and you will soon see the effects of disrupting our microbiota.
There is much interest in the microbiota that inhabit our bodies. For instance several studies have indicated that intestinal microbes can play a big part in obesity, with changes in the makeup of the microbiota being a major risk factor (1). But many of these organisms are hard to learn about—the ones that inhabit the deep folds of our gut thrive in moist, warm, anaerobic conditions with lots of specialized nutrients, conditions that are very hard to replicate in the laboratory. For that reason, we don’t know much about many of the microbes that are the most abundant within us.
The Human Microbiome Project begun in 2008 by the National Institutes of Health (2) seeks to understand human microbiota and their relationship to human health. To do this, the researchers leading the project took a metagenomic approach—using advanced DNA sequencing technologies to sequence the genomes of human microbiota and get a look at the human microbiome—without culturing the microbes.
But to truly understand their biology, and to perhaps exploit what we learn to enhance human health we need to be able to manipulate these organisms. In particular, biologists who are interested in synthetic biology would like to use these micro-organisms to monitor what is going on in our bodies, particularly our guts. What better monitor for these hard-to-access places than an organism that is already well adapted to live there? Continue reading →