This blog was written with much guidance from Jennifer Romanin, Senior Director IVD Operations and Global Service and Support, and Ron Wheeler, Senior Director, Quality Assurance and Regulatory Affairs at Promega.
A Trip Down Memory Lane
Back in the day when we all walked two miles uphill in the snow to get to our laboratories, RNA and DNA extraction was a home-brew experience. You made your own buffers, prepped your own columns and spent hours lysing cells, centrifuging samples, and collecting that fluorescing, ethidium bromide-stained band of RNA in the dark room from a tube suspended over a UV box. Just like master beer brewers tweak their protocols to produce better brews, you could tweak your methodology and become a “master isolater” of RNA. You might get mostly consistent results, but there was no guarantee that your protocol would work as well in the hands of a novice.
Enter the biotechnology companies with RNA and DNA isolation kits—kits and columns manufactured under highly controlled conditions delivering higher quality and reproducibility than your home-brew method. These systems have enabled us to design ever more sensitive downstream assays–assays that rely on high-quality input DNA and RNA, like RT-qPCR assays that can detect the presence of a specific RNA molecule on a swab containing only a few hundred cells. With these assays, contaminants from a home-brew isolation could result in false positives or false negatives or simply confused results. Reagents manufactured with pre-approved standard protocols in a highly controlled environment are critical for ultra sensitive tests and assays like the ones used to detect SARS-CoV-2 (the virus that causes COVID-19).
The Science of Manufacturing Tools for Scientists
There are several criteria that must be met if you are
producing systems that will be sent to different laboratories, used by
different people with variable skill sets, yet yield results that can be
compared from lab to lab.
These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.
While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users:
Tailing blunt-ended DNA fragments with TaqDNA Polymerase allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors. This method also eliminates some of the requirements of conventional blunt-end cloning — Fewer steps, who can argue with that?
Today’s blog is jointly written by guest blogger Peter Kritsch, Biotechnology and Biology Teacher at Oregon High School and contributor Barbara Bielec. K-12 Program Director at the BTC Institute.
The BTC Institute has offered two graduate-level courses for high school teachers for many summers. Biotechnology: The Basics and Biotechnology: Beyond the Basics have become very popular and are also drawing the interest of middle school teachers. So, this June we piloted a new 3-day course designed specifically for them. Representing different schools and districts, eight teachers learned how to extract DNA from strawberries, pour and run agarose gels, identify a taste gene, and received information on lots of resources to use with their students.
Through the BTC Institute’s Biotechnology Teacher Academy, these courses are offered at no cost and $300-$500 stipends are available. A main Academy goal is to provide high quality professional development opportunities that prioritize content that participants can smoothly incorporate into their classrooms. Our commitment to stipends is generously supported by the Wisconsin Space Grant Consortium (WSGC), Promega Corporation, Madison College and the BTC Institute. (All three courses are offered for graduate credits from Edgewood College, and Viterbo University also offers credits for the two geared to high school teachers.)
The importance of this approach is affirmed by Sherry Jacobsen (Kromrey Middle School in Middleton, WI):
This [course] is such a gift to teachers! Many times we aren’t treated as professionals so it was nice to be treated as a professional without a high personal cost. I love how the course is so practical. Many courses are only in theory and no application. I can take so many useful ideas with me.
Biotechnology is making its way into the middle school classroom. With access to the BTC Institute’s Equipment Loan Program, teachers can check out micropipettes, gel boxes & power supplies, an ultraviolet light box and other equipment for up to two weeks. Course participant Amy Reimer (Core Knowledge Middle School in Verona, WI), has already taken advantage of this program and noted that it was “great to review procedures” through the course and plans to borrow equipment again this coming year. Continue reading “A Successful Launch for Biotechnology: The Basics for Middle School Teachers”
The University of Wisconsin Master of Science in Biotechnology Program began with its first cohort of students in 2002, and its 14th class graduated this May, with the BTC Institute serving as a major partner since its inception. The 15-year anniversary highlights the success the program has garnered over the years, with over 300 alumni successfully completing the program between 2002 and 2017.
The On the Road (OTR) BTC Institute Biotechnology Field Trips (BFT) program is rolling right along! We are doing our best to brave the winter weather to take hands-on science activities all over the state of Wisconsin.
The BTC Institute BFT program served over 3,400 students last year, most of them here at the BTC in Fitchburg. That said, each year the OTR part of the program is growing in order to serve schools that cannot travel here for various reasons, such as distance, bus costs and the need to minimize out of school time. Continue reading “On the Road with the Biotechnology Field Trips Program”
When you are faced with a new research challenge or are troubleshooting in the lab, nothing replaces the wisdom of the lab tech down the hall who has 20 years experience doing the very technique you need to try.
But, sometimes there isn’t a local expert handy.
The Citations Database on the Promega website provides another source of expertise for you. We curate peer-reviewed publications that cite the use of Promega products so that you can see what people have done and how they have done it. We include links to PubMed for articles that are in indexed journals, and we also include brief notes about how the Promega products were used in the research.
Antibiotic-resistant bacteria and their potential to cause epidemics with no viable treatment options have been in the news a lot. These “superbugs,” which have acquired genes giving them resistance to common and so-called “last resort” antibiotics, are a huge concern as effective treatment options dwindle. Less attention has been given to an infection that is not just impervious to antibiotics, but is actually enabled by them.
Clostridium difficile Infection (CDI) is one of the most common healthcare-associated infections and a significant global healthcare problem. Clostridium difficile (C. diff), a Gram-positive anaerobic bacterium, is the source of the infection. C. diff spores are very resilient to environmental stressors, such as pH, temperature and even antibiotics, and can be found pretty much everywhere around us, including on most of the food we eat. Ingesting the spores does not usually lead to infection inside the body without also being exposed to antibiotics.
Individuals taking antibiotics are 7-10 times more likely to acquire a CDI. Antibiotics disrupt the normal flora of the intestine, allowing C. diff to compete for resources and flourish. Once exposed to the anaerobic conditions of the human gut, these spores germinate into active cells that embed into the tissue lining the colon. The bacteria are then able to produce the toxins that can cause disease and result in severe damage, or even death. Continue reading “Shining Light on a Superbug”
BTCI provides our students an opportunity that they could never get in the classroom. —Jim Geoffrey, Biology Teacher, Kaukauna High School
Your bus has arrived and parked in the circular driveway at the front of the BioPharmaceutical Technology Center on the Promega Corporation campus in Fitchburg, WI. Your BTC Institute hosts – and instructors – for your field trip are Barbara Bielec (K-12 Program Director) and Ryan Olson (Biotechnology Instructor). They’ll greet you in the Atrium and direct you to a conference room where you can leave coats and backpacks, and then to the lab you’ll be working in during your visit.
Here’s a taste of what happened next for students from Random Lake High School and Wonewoc High School on December 3rd, and from Kaukauna High School on December 4th.
For three out of the last four years, we have been honored to have one of our key technologies named a Top 10 Innovation by The Scientist. This year the innovative NanoBiT™ Assay (NanoLuc® Binary Technology) received the recognition. NanoBiT™ is a structural complementation reporter based on NanoLuc® Luciferase, a small, bright luciferase derived from the deep sea shrimp Oplophorus gracilirostris.
Using plasmids that encode the NanoBiT complementation reporter, you can make fusion proteins to “report” on protein interactions that you are studying. One of the target proteins is fused to the 18kDa subunit; the other to the 11 amino acid subunit. The NanoBiT™ subunits are stable, exhibiting low self-affinity, but produce an ultra-bright signal upon association. So, if your target proteins interact, the two subunits are brought close enough to each other to associate and produce a luminescent signal. The strong signal and low background associated with a luminescent system, and the small size of the complementation reporter, all help the NanoBiT™ assay overcome the limitations associated with traditional methods for studying protein interactions.
The small size reduces the chances of steric interference with protein interactions. The ultra bright signal, means that even interactions among proteins present in very low amounts can be detected and quantified–without over-expressing large quantities of non-native fusion proteins and potentially disrupting the normal cellular environment. And the NanoBiT™ assay can be performed in real time, in live cells.
The NanoBiT™ assay is already being deployed in laboratories to help advance understanding of fundamental cell biology. You can see how one researcher is already taking full advantage of this innovative technology in the video embedded below:
Visit the Promega web site to see more examples more examples how the NanoBiT™ assay can break through the traditional limitations for studying protein interactions in cells.
You can read the Top 10 article in The Scientisthere.