An Inquiry Driven Visualizing DNA Using Fruit

My goal for this year is to pop the hood on my teaching pedagogy and write about how to practically teach molecular biology. Over the next few months, I am going to be posting information about teaching common biological activities but in a more intuitive that makes the procedure less cookbook and more of an exploration. The first entry is about the very common practice of purifying DNA from fruit using simply household chemicals, a great starting point for the practice of molecular bio. Many students have done this but there are many different adaptions and add-ons one can do to the procedure in order to add elements of experimentation and mystery all while exploring the chemical properties of the DNA molecule.

Students tend to vary greatly in their overall knowledge of DNA and so it is important to ensure students understand what it is and provide a number of fast facts that will hook the student’s attention.

Talking Points to Build Interest Around DNA Prior to Purifying It

  • Each one of your cells has about 6 feet of DNA packaged inside. DNA is coiled around a complex of proteins called histones which tightly bond to the DNA and keep it condensed. Since this is an abstract concept to envision, envision a long of yarn wrapped into a tight ball. Unwrapped, it would stretch on for a hundred foot but when wrapped, it is easy to handle and takes up a lot less space
  • If one were to remove the DNA in each of their cells and line up the strands from end to end, it would stretch to somewhere between Jupiter and Saturn! Imagine spending months upon months in a space ship viewing the same person’s DNA the whole time. Wow!
  • Most of the attention DNA gets is from genes, the blueprints for proteins that students and scientists alike spend lots of time studying but that only accounts for a small percentage of the overall genome! The other sequences, called introns, have a fascinating story to tell as well.
    • Transposons are remnants of viruses that have implanted themselves in our DNA and are present in our evolutionary ancestors. During times of extreme cellular stress, some have the availability to catalyze their own removal and move to different places in the genome.
    • There are also psuedogenes, inactive copies of nearby genes that collect mutations at a higher rate than their functioning neighbors.
    • There are sequences that allow for intramolecular bonding that can be sometimes be millions of bases apart but loop together to find each other to allow for tighter packing. After all, 6 feet of DNA have to fit into a space far smaller than the head of a pin

Materials

Quart freezer bags
Fruit (strawberry works best but bananas and blueberries also work)
Shampoo
Salt
Meat Tenderizer
Soap
Plastic cups or beakers
wood or glass stir rod
Cold 99% Isopropyl Alcohol or Ethanol

Note: The most important piece of pre-experimental planning is to place the alcohol required for the last step of the process in a freezer. The DNA solubilizes in alcohol and chilling the alcohol enables the DNA to clump together faster as well as rise to the top.

Below is a common procedure for purifying DNA from fruit. Connections to other areas or experimental variations are in bold below the steps. 

  1. Remove the stem and place the strawberry in the bag and seal, ensuring that all the air has been removed from the bag

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  • Mathematics connection: Have the students weigh the strawberry and hypothesize what percentage of the total weight of it is represented by DNA.

2. Gently mush the strawberry ensuring that all chunks are broken into small pieces. No single piece of strawberry is larger than a couple of millimeters

  • Math/Physics connection: Why is it important to get the pieces as small as possible? Take a rough volume of the pieces and consider the importance of surface area in allowing reagents to reach their target. 

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3. Place 1 squirt of soap/shampoo along with 1/2 tsp salt or meat tenderizer into the bag and 25-50 mL of water into the bag and gently slosh around the slurry allowing the surfactants and

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  • Experimental design challenge: What effect would changing the amounts of  reagents have on the amount of DNA recovery? Students can change one variable at a time and see how that changes the overall DNA yield
  • Chemistry connection: Soap is required to break down the cell membrane, which is largely composed of lipids. Why is soap such a powerful degreasing agent? And why don’t our cells pop open when we use soap?
  • Biochemistry connection: Why is meat tenderizer used? Read the ingredient label and hypothesize which components of the tenderizer make the most different. 

    4. Allow the bag to rest for 10-15 minutes to allow the chemical reactions to proceed

5. Pour the contents of the bag into a coffee filter place on top of a beaker and allow the liquid containing the DNA to filter down into the beaker below. 

6. With the DNA solution in the beaker below, about 2-3 volumes of  COLD alcohol can be poured in. The results work best if the students are stirring their solutions as the alcohol is poured.

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The DNA should be stuck to the stir rod and the students can squeeze out as much of the water and alcohol as possible. If the students weighed the strawberry in the first step, they can weigh the DNA, on the stir rod and calculate what percentage of the total weight of the strawberry is taken up by the genetic material. 

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This process can be repeated with different fruits though their DNA is not as sticky as strawberries. This can make quantitative comparisons of fruit DNA more difficult but a qualitative observation is still possible.

 

History Connections to DNA in general

  • The discovery of DNA as the genetic material of the cell and its structure was filled with colorful scientists and characters. The research of Griffith, Avery, McCleod, Watson, Crick, and Franklin helped to prove the properties and structure of DNA. Their research and lives also demonstrated what science in the 1940’s and 1950’s ws like. The discovery of the double helix was a race between competing labs and while Watson and Crick published first, there were others. What were some of the other researchers studying DNA and what contributions to science did they provide?
  • While Watson and Crick get most of the credit for the discovery of the DNA double helix, Franklin’s work on elucidating its X-ray structure was paramount. For decades, her work was marginalized. What were the social circumstances around the role of women in science during that era and how has it changed?
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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.  

 

The Quest to Cut Vocab and Not Experience

I went to school for molecular biology for 6 years and then worked in the field another 7, so how do I distill down 13 years of jargon so that my middle schoolers will be able to do a transformation and understand the general process and how it goes down? I start with useful vocab that they need to be familiar with so they understand the procedure: competent cells, transformation, DNA, and a few others. Then, the reagents: Inoue Buffer, E Coli, ampicillin, plasmid. This way, the students can read through the manufacturer’s protocols and follow the procedure as well as understand the basic idea of how DNA works in concert with organisms. There are definitely details I leave out, but I can guide students toward them in post-lab or focus on them in other activities.

Apparently, there is a name for this. It’s called “just in time” learning, and it reminded me of the sort of thing I had to do in the workplace. A problem would arise regarding a certain pathway. Then, I had to research and understand the pathway using literature and find a way to problem solve by utilizing experimental design. Of course,” just in time learning” isn’t just limited to the sciences. It is useful to every field, and I love lessons where science can be used as a jumping off point into multidisciplinary areas. After all, no one concept is an island.

The key terms that I chose to concentrate on I deemed to be a reasonable entry point for the students who have some knowledge of general biology but lack a lot of the finer details. I planned a follow up lesson using DNA ligations to explore more about DNA, including some gritty details of its structure, such as the directionality of the backbone, an essential concept in understanding restriction enzymes, their mechanism, and their usefulness when working with DNA plasmids. By the end of that unit, my hope is that students will have worked with DNA enough so that not only the concept of genes, but of all of the things that work with things to make proteins, is emblazoned in their minds.

As the student’s lab reports are coming in, I will see how well they integrated the knowledge. The more advanced the students are, the more I expect to see ties into that theory, and my revisions for them will probe into the areas where the detail was scant or off. Making mistakes is the first step to fluency whenever one learns a new language, and molecular biology is just that.

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The Importance of Food Labs

Kids love to eat, and they obviously love food, so it stands to reason that they love labs in which they can eat what they produce. After all, any science teacher can tell you that kids ask if they can eat lab materials ALL THE TIME. It doesn’t matter if it’s sugar or sulfuric acid, as long as it looks edible, the questions will fly. That is why it’s important to include food labs in the class repertoire and finally be able to answer “yes” when a student asks if they can eat a lab material.

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In response to the fact that we had been studying bacteria and how it grows, a student asked if we could make yogurt. Given that bacteria factor heavily into the yogurt production process, I realized this would be a great way to continue our exploration. We already saw the effects of bad bacteria, so why not focus on the useful parts of this gigantic kingdom of microscope creatures? After a very thorough cleaning of the lab and incubator using UV lights to ensure sanitary conditions, we began our deep dive into food production.

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We started off simply by using yogurt (greek or regular) as a base to grow in milk (whole, 1%, 2%, chocolate). The students realized quickly that the bacteria in the yogurt is going to reproduce at a pretty rapid rate, consuming the milk as it goes and doing whatever it does to produce the stuff that they know well and eat on a regular basis. One important experimental note I told them was that they had to boil their clean, newly purchased, “food safe,” beakers prior to each bacterial growth, as well as heating the milk up to 85C-90C. I didn’t expressly tell them it is to kill off bad bacteria; they looked that up for the post lab questions. Also, some students made the mistake of putting their yogurt in immediately after heating and were disappointed to discover that no growth had occurred within the yogurt. They quickly realized that they needed to cool the milk down so as to not kill the newly added bacteria, which was a valuable part of the scientific process.

For the follow up experiments, we looked at using a variety of different yogurt starters, each with slightly different bacterial compositions. The students researched the different strains to determine what the packets had it common and hypothesized the exact amounts of bacteria in each one. They performed a number of growth cycles looking at the effect of these starters and how they affected taste, consistency, and speed of growth of the yogurt, perfecting their protocols at each step.

Abby getting ready to consume said yogurt

Abby getting ready to consume said yogurt

I made sure not to give them too much of the story behind why yogurt is formed and allowed them to uncover the science behind it after the experiments. When we concluded the yogurt cycles, the students had a lot of questions as to what they had just done. I guided them toward a list of basic questions, which included, “What about the bacteria makes milk thicken when incubated?” The students uncovered pretty quickly that the bacteria eat the milk sugar (aka lactose) for food and produce lactic acid as a waste product, which changes the pH of the milk, degrading some of the proteins, and thus thickening the overall product. This is similar to the effect of acid on enzymes and goes along with the general acid-base studies we did earlier in the year, so we were able to use this as a summative exercise on important concepts we’ve covered throughout the school year. While I could say that this was my plan all along, I will admit it ended up being a very happy accident.

Next, we explored the production of cheese, which was also suggested by a student. We designed two basic types of cheeses: acid and rennet produced. In addition, we used a whole new set of bacterial starters designed for cheese. We started with acid-formed cheeses that use the same coagulation principles as yogurt to curdle milk, but under high heat the curds (mostly fats and protein) split from the whey (mostly sugar and some protein), which can then be strained to produce anything from farmer’s cheese to mozzarella. Rennet is an enzyme solution that breaks down lipid and protein structures in the milk resulting in many common aged cheeses, like cheddar, and ties together some concepts from our enzyme unit earlier in the year.

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We spent about 3 weeks exploring the production of cheese and looking at it from various scientific angles to see if the students could get a smoother texture, stretchier mozzarella, and all around tastier cheese by modulating the flavors with salt and other spices. Since most of the students were using recipes they found online, they also got practice in following directions and altering steps when time was a problem to see if that changed the overall structure of the cheese. By the end, many students were creating their protocols based on prior research, which really is the heart of proper experimental design.

The principles used to make the two foods are similar, and the students really enjoyed the exploration and the act of producing something at the end that was edible.  They also received important knowledge as to what is in their food and how science is used to create the things they like. I sampled most of the students’ work and suffice it to say that many of the creations were quite tasty. So not only can food labs be a treat for kids, but instructors as well, and since they all followed proper safety protocols, not one illness resulted. Overall, this was a great summative exercise to tie together many of the previous concepts we have studied over the past year.

In celebration of Mystery in Science

As many teachers do during the summer, I brainstormed some ideas and themes I wanted to discuss in the upcoming school year. To do this, I started comparing my experience teaching science with that of my life as  a researcher in R&D. I concluded that there was an element of mystery that drove my bench research that was decidedly missing in many science classrooms. For bench scientists, much of their time conducting research is spent controlling variables and perfecting models that seek to reduce as much mystery as possible but undoubtedly, some still remains. For students, that is a good thing. When mystery is properly integrated, it facilitates curiosity, a necessary skill for all students to have regardless of the subject matter.

Most labs in science classrooms are designed to reduce frustration associated with the mystery of lab work. The steps are clear or can be fairly easily elucidated, and the results are usually prescribed given that post lab questions are often tied to the achievements of a certain result. This predictability can  be easier for the student, especially those who aren’t comfortable with the idea of “not getting the right result.” This summer, I have been thinking about ways to resolve the differences between the true nature of mystery in science with the student’s desire for scientific predictability. This blog entry will not provide a decisive answer but rather highlight some of the places I am going to add elements of mystery to my lab curricula this year.

I am calling the first type “Mystery in Method.” The students start with the same set of ingredients and devise a way to get to a set end point. Example experiments could include giving each student a plant in water (elodea or chara) and creating a methodology to increase and/or decrease photosynthetic rate by altering the water environment. Obviously, the mystery is creating a testable protocol to follow to reach a conclusion which, in this case, would be some readout of photosynthetic efficacy, perhaps CO2 consumption or O2 production. I’ve already integrated Mystery in Method many times in public schools and noted that while there was initial hesitation and lots of frustration, with guidance and time, the students adapted to it and found a greater sense of engagement in lab by year’s end.

The second type is called “Mystery in Results”, and obviously the mystery appears at the end. This can usually be accomplished by giving the students a specific set of instructions to follow to look at some experimental read out of their experiment. One example would involve students growing bacteria from different sources and isolating their DNA. Then, the students could perform a restriction enzyme digest on their own samples to see how the sequences of the DNA differed by running the purified DNA samples on an agarose gel to examine the different lengths that result. Both the digest and the gel have their own specific instructions that must be followed, but the starting and ending points of the experiment allow for an element of mystery. Another example would be doing a simple chemiluminescence experiment with luminol, ammonium ferracyanide, hydrochloric acid, and sodium hydroxide to get a glowing beaker. Here the students don’t know what the end looks like but will get a truly cool looking result if they follow the steps accurately.

The final type of experiment is what I refer to as the “Cave of Mystery” and combines the creation of a protocol with the gathering of mysterious results. This experiment is the ultimate replicator of mystery as experienced by biotechnological researchers. It begins with an idea and not much else. It’s then up to the student to determine how to research the idea, devise a strategy to test it, determine what data to collect, and how to use what is collected to guide further experiments. This sort of experiment is challenging, time-consuming, often repetitive and ultimately, the best way to understand true scientific inquiry. I’ll write a longer post about navigating the “Cave of Mystery” as a means of science fair project creation during the winter, but for now these sorts of experiments are great long term projects.

By re-introducing science students to the power of mystery, it creates ownership of the experiments and really gives the students a chance to experience the ectasy and agony of being a scientist. So here’s to a wonderful 2015-2016 school year, and may you all embrace the mystery of discovery!