In Celebration of “Why Not?”

This year has been all about molecular biology,  studying its possibilities and how scientists have used simple organisms to perform work on a cellular level. In November, we performed bacterial transformations where we shoved a plasmid into E Coli that enabled it to change color. Then, we performed restriction enzyme digests on a plasmid and inserted a gene that changed its color and enabled it to grow on antibiotic resistant media.  I gave the students an article on CRISPR prior to our last round of experiments to peak their interest in the potential of gene therapy. Fast forward a few months later when a new collaboration will have us doing CRISPR experiments in lab.

 

I was recently in a meeting with our outreach director talking about this project, and she asked me why I’m doing this.  I simply replied, “Why not?” But allow me to back up.

 

CRISPR is a breakthrough technology that is essentially a set of DNA scissors that can be directed to cut genes at certain points. CRISPR (which stands for Clustered Regularly Interspersed Short Palindromic Repeats) utilizes an enzyme called Cas9 that was originally found in bacteria to make the cuts and offers a potential opportunity to edit genes in vivo, enabling scientists to remove harmful genes that could at some point cause diseases like cancer. There are obviously a host of ethical and legal debates that can stem from this technology, but we will put those aside for now and focus on the project we are doing in lab.

 

Much of molecular biology in a lab setting involves utilizing organic macromolecules to perform work in cells, enabling scientists to see the effect that altering and manipulating pathways have on various levels of the cell (global and/or local). Practically, this gets done by adding small volumes of liquid containing sensitive reagents to other small volumes of liquid containing different sensitive reagents. The nice thing about these experiments is that if  students have performed one of these assays, they can basically perform a majority of all molecular biology assays, including CRISPR.

 

CRISPR is arguably the hottest area of biology right now. Do a google search for it and a litany of articles will pop up from a variety of non-industry sources. It seems that any news source worth their clicks has had an article detailing the many cool aspects of this emergent technology. Further, there is a nifty beginner CRISPR kit available on Odin, a great website/store for amateur scientists that sells an inexpensive molecular biology kit with the real reagents.

 

So, given its popularity, and given that my students have already done similar experiments, why not do this in the classroom?

 

For me, this is the fun part of doing deep dives into a given content area. By spending a while in a given field, it provides the opportunities to build the background necessary to do experiments that are going on in labs right now. Restriction enzymes were an extremely hot advancement back in the late 60’s,  and it took nearly 3 decades for them to appear in high school classrooms. With the products currently available and with the proper knowledge base, it is possible to have students learn about something that scientists are feverishly studying right now, which consistently excites curious minds.  Students are inevitably more engaged when they realize that the projects they are working on have concrete links to real world phenomena.

 

Since my students only meet with me for 2 hours a week, it has taken a while to get to this point, but it is quite feasible that a student can go from extracting DNA from strawberries to CRISPR in a matter of 4-6 weeks. If you are interested in learning how, I will be posting the unit plan summaries over the course of the next couple months.  

 

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Do Spices inhibit bacterial growth?

There are so many ways in which studying bacteria is useful from an educational standpoint.

It enables students to envision the lives and activities of the smallest and most ubiquitous forms of life on Earth and relate their activities to their own. All bacteria survive by getting resources, reproducing, and warding off prey. They also respond to stimuli, have a penchant for certain foods, and in our own bodies, outnumber our cells. Some are dangerous, some are innocuous, and all are mysterious given their microscopic size.

We started our adventure learning about bacteria by doing a quick web search to return general facts about them. I gave the students 20 minutes to uncover as many things as possible about them and then created a master list of bacterial information. This included everything about reproduction to hand sanitizers to their ability to survive in space under the right conditions. After that, we began our first experiment by thinking about spices and the original purpose of spices in preserving food. I purchased 4 spices: cayenne, cumin, paprika, and black pepper. I gave the students a simple experimental goal and asked them to design an experiment to determine if spices inhibit bacterial growth.

I gave them petri dishes, glassware, agar, spices, and nothing else as the students looked up how to prepare the plates for the bacteria to grow. It was interesting for me to watch them struggle with things that as a scientist seemed second nature – like how to dissolve the agar, how much of it to use, and when to apply the spices (in the agar directly vs. sprinkled on top). What resulted was a wide range of plates with different combinations of agar andspices. As a whole, they were all curious as to whether or not bacteria would grow and what it would look like. Several days later when they returned to lab, they saw that bacteria was growing on practically all the plates which disagreed with most of their ideas regarding how spices should inhibit bacterial growth. Some even saw growth on cayenne and cumin that looked like mold. As I told them, scientists can’t just make assumptions and that more analysis needs to be done, so we saved those plants with the hope of purifying DNA and sending it out for sequencing.

For their follow up experiment, I wanted to them to mutate their bacteria. There are a great many ties to some very socially relevant problems regarding bacteria and drug resistance. I shared with them the case study of tuberculosis in Russia as a particularly dangerous example (http://www.nature.com/news/russia-s-drug-resistant-tb-spreading-more-easily-1.14589). For this, I wanted them to take bacteria from their spice plates and replate it with various substances added that might confer resistance, such as ethanol.

We’ve discussed ethanol in various capacities regarding its antiseptic abilities. They all know that it and isopropanol are used in products like Purell that boast of killing 99.99% of bacteria. We discussed what that actually means and that if a trillion bacteria exist (a likely scenario for most surfaces), there are still millions that survive possibly with something genetic that will enable them to propagate back to their original numbers within a matter of days or even hours. Indeed the students saw that ethanol didn’t seem to inhibit growth when stretched out for a long period of time and in a couple cases, encouraged growth. This was a truly fascinating thing to witness and taught them a valuable lesson regarding the life cycle and adaptability of bacteria.

Our final experiment consisted of our first steps in classifying bacteria with a simple gram stain. This tests for the presence of a carbohydrate called lipopolysaccharide, a molecule that helps bacteria to resist antibiotics. Some strains have it and others do not and through a series of dyes, the LPS containing bacteria are revealed colorimetrically. For the first time in this series of tests, I gave them an exact protocol. At first glance, following protocols seems counterintuitive to creativity but the ability to follow steps and get a result is extremely important to life. After all, when one is putting together furniture, it’s probably not a good idea to skip or veer from the steps. In addition, getting a result is not the same as getting a prescribed one and most students saw mostly gram positive but also several colonies of gram negative. This showed students that different types of bacteria were growing in their cultures. Ideally, we would have loved to sequence all the bacteria but unfortunately, DNA sequencing is still an expensive endeavor when done with many samples.

This series of experiments provided a valuable introduction to bacteria, their life cycles, and ability to survive and thrive in a variety of conditions. By doing this, we took the microscopic and brought it out to be observed by the naked eye so that we may truly see life on the smallest scales up close.