Experiments

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I have used the experiments described below when teaching introductory Molecular Biology in a college-level course for advanced high school students. Some of these experiments were developed by others and some were partially or fully designed by me. I have provided links to experiments designed by others. Feel free to modify the experiments that I designed for your own use and distribute copies to your students (with citation), but please do not distribute these publicly. There are many similar lab experiments that could serve the same role as these, some of which are sold as kits. I have not used any kits myself, so cannot comment on particular vendors. When my classes have been large, I have students work in pairs. When my classes have been 10 or fewer students, I have students work alone. Working alone is definitely ideal because students become truly proficient. However, working in pairs is much easier to manage in terms of the partners compensating when students are absent. 

Unit 1: Introduction to Biochemistry and Cell Biology

Unknown Organic Molecules

  • After a lesson on pipetting and sterile technique, students conduct a lab where they need to determine the identity of the solutions in several tubes, each of which contains one or two types of biological molecule. Students are asked to practice sterile technique even though it doesn't matter yet in this lab so that they can practice for when it does matter. The logic portion of the lab tends to be straightforward for most students. My main goals are for the students to practice pipetting, practice sterile technique, obtain experience with microliter quantities, and to experience using the equipment available in the lab. This is also a first experience where students can think about including positive and negative controls. There are also not explicit labeling instructions, so students start to get used to having to label tubes in a meaningful way. This is the protocol I have used based on the equipment we have, which could be adapted for other labs. I have tended to have students do all of the tests except the gel in a 90 minute period, followed by running relevant samples on a gel the next class in a 45 minute period. I have them run samples on gels even if they already think they've figured it out so that they can experience gel electrophoresis and confirm their hypotheses. For gel electrophoresis, I routinely have students use sodium boric acid buffer (Brody and Kern, Biotechniques 2004) in teaching labs because it can tolerate a very high voltage which facilitates students having the time to load, run, and photograph gels within a single class period. We use Biotium's GelGreen for DNA staining due to its high sensitivity and the danger of using the mutagen ethidium bromide.

Biofuels

  • There are many published and commercially-available lab kits related to biofuel production that could be substituted for the wet lab describe here. I have students set up yeast fermentation reactions in plugged 12mL luer-lock syringes and measure the volume of carbon dioxide produced. I wash and reuse the syringes and caps each year. 
    • [I've also used tiny test tubes inverted in medium test tubes to qualitatively study fermentation, but it requires large volumes and tends to produce much more gas than the tiny test tube can hold. There's an intriguing protocol that I haven't tried yet using microcentrifuge tubes to quantitatively study fermentation in The American Biology Teacher 79, 414-420 (2012)]. 
    • I provide a variety of carbohydrate starting materials and a variety of enzymes that might convert carbohydrate polymers into glucose. These include commercially-available amylase and cellulase, as well as specialized cellulases that I requested as free samples from DuPont. I buy packaged dry yeast from the grocery. When we've used newspaper as a starting cellulose material, the solutions have started to stink and I think this may be due to bacterial contamination. 
    • I allow students to design their own experiments, with 2-3 pairs of students collaborating on experimental designs and splitting the setups between them. I don't give a lot of boundaries for their experimental design, but I do remind them that they should include appropriate controls. 
    • The experiments tend to be very messy in several respects: some of the carbohydrate solutions are chunky, so it's difficult to pipet them appropriately; I haven't found a great way to pretreat cellulose in a safe way at a school, although I've had limited success with the alkaline/peroxide method that's described; grocery store-bought yeast can yield some carbon dioxide when only combined with water (maybe they can ferment the chemicals they're packaged with?) so a yeast-only control is critical; and students nearly always omit controls that would be necessary to analyze the data. 
    • However, despite this messiness, I like this as an early experiment in the course because 1) it leads to good discussions about the appropriate use of positive and negative controls, 2) it gives students an opportunity to remember the mole conversion and dilution calculation skills they had learned in Chemistry, 3) it highlights the role of enzymes and conditions for their optimal activity, and 4) it gives them some perspective on the real-world difficulties of not only industrially producing biofuels, but also of conducting experiments where answers are not predetermined.
    • Biofuels Lab student handout and analysis questions
    • Biofuels Lab Sample Calculation Worksheets
  • Following the wet lab, I have my students analyze the considerations for the development of better biofuels (questions included in analysis section of student handout) by analyzing data in the following sources:

Unit 2: Protein Structure and Function

3D Molecular Designs Map of the Human Beta Globin Gene Hands-On Activity

  • I highly recommend this activity as a way for students to discover codons, stop codons, introns and exons for themselves. I do it early in Unit 2, preferably before students have read about transcription and translation.

3D Molecular Designs Amino Acid Starter Kit Hands-On Activity

  • The Amino Acid Starter Kit is a great hands-on way for students to learn the principles of protein folding. This is one of the most important activities that I do in my SMART Team protein modeling classes and I also tend to use it in my Molecular Biology course.


Fluorescent Protein Analysis

  • Princeton University's HHMI Molecular Biology Outreach Program developed an Investigation of Fluorescent Proteins. In this laboratory, students start by transforming one of two mixtures of three fluorescent protein plasmids into JM109 bacteria. This is great for introducing the basics of central dogma and transformation. The fluorescence is definitely much brighter in JM109 (which should be used) than in DH10beta or DH5alpha, which seem to have a more problematic time handling the protein overexpression.
  • In the second part of the Princeton laboratory, students spread bacteria from one colony onto a fresh plate that will be used for purifying the fluorescent proteins. At this stage, it's best for future experiments if students choose GFP, YFP, mCherry, or mTangerine. BFP can also be used, but it's more difficult since it can only be seen under UV light. mGrape should not be chosen since its fluorescence is too dim to be observed. In practice, when doing this step, I've found that it sometimes works well, with plates that are completely brightly-colored. However, sometimes the color doesn't develop and I'm not entirely sure why, but might have to do with the density of bacteria - with more dense producing less color. I generally spread about twice as many plates as we should need to ensure enough good ones are made. Students also tend to have a hard time understanding what it means to spread bacteria completely over the whole plate, which leads to more plates that aren't ideal. Sometimes at the end of the purification, there is just a glob of goo and not much supernatant. In this case, add an extra 500µL of 1X TE, mix and centrifuge again for at least 5 minutes. After this, the fluorescent protein should be in the supernatant and the pellet should be mostly white. This method should also work if there's still a lot of color in the pellet.
  • In the third part of the Princeton lab, students use purified protein solutions to compare how different proteins can be denatured by acid, base, detergent, and heat. If students compare fluorescent proteins that are point mutants of each other (ie BFP, GFP, YFP), then questions can be asked about how point mutations can affect protein structure.  I use heat blocks set to 80°C and 100 °C and solutions of 0.25M HCl, 0.25M NaOH and 10% SDS that can be added to fluorescent protein samples.
  • In the fourth part of the Princeton lab, students run fluorescent proteins out on agarose gels. Samples of fluorescent proteins should be saved for this before starting the above denaturation studies. The protocol calls for ultra pure agarose, but I just used general purpose agarose. Examples of data from these gels are shown here and an inquiry-approach to analyzing the data is in Class Activity 2B: Fluorescent Protein Analysis in Molecular Biology Concepts for Inquiry: The Exploration Workbook.
  • A more refined purification of the fluorescent protein can be performed using nickel beads to capture the His tag on the fluorescent protein as described in the UCSD ScienceBridge Fluorescent Protein Purification Protocol. I've created a version of this using Promega's His-Link spin columns which is here. Sometimes, color doesn't develop well in the liquid cultures - scraping up some fluorescent bacteria from a plate to start the purification works fine and might be a preferable way to do this. The fluorescent protein expression is so high that that there is often extra in the flow-through.
  • Purified fluorescent proteins were then digested with proteases in an experiment inspired by the protease portion of a GFP laboratory developed at the University of Massachusetts that used meat tenderizer (papain). My version using pepsin and papain and the analysis of the digestion on a polyacrylamide gel and with ninhydrin assays is here. Note that the elution buffer from the His-link spin columns is definitely a buffer, and a substantial concentration of HCl must be added to overcome its buffering capacity to allow for pepsin digestion. The molarity of the resulting mixture is so high that the sample runs somewhat abnormally on the polyacrylamide gel.

Unit 3: DNA Replication, Repair and Genetic Engineering

Detection of Genetically Modified Food Through PCR

  • I use this lab as the students' first experience with PCR. I've found that corn-based products work well, but wheat-based products do not. It's easier to get a DNA prep that can be PCR'd by using leaves rather than fruit. Students should consider whether their source contains a part of a plant that would contain DNA. For plastic microcentrifuge tube pestles, just washing the pestles in water and reusing the pestles is not sufficient to remove residual DNA. Pestles need to be soaked in 10% bleach for several minutes and then washed with a lot of water in order to be reused. I've also tried making my own pestles by molding a dollop of hot glue onto the end of a 1000µL pipet tip and that works well enough, but is time-consuming for materials that can't be reused.


Gibson Assembly Mutagenesis of Fluorescent Protein Genes

  • A student lab activity to mutate GFP into BFP or YFP was developed in the UCSD ScienceBridge Socrates Fellows program by Graduate Fellow Alyssa Wu Zhang and Teacher Partner Jesse Wade Robinson. I have had students use more than one method to mutate BFP, GFP, YFP, mTangerine, or mCherry over the years, but in recent years I have settled on a variation of Gibson assembly in order to introduce one or more mutations into these genes. Gibson assembly with mutagenesis has been more effective than other methods I have tried with students and has the advantage of allowing multiple mutations to be introduced at one time. The more mutations that are introduced, and therefore the more fragments to assemble, the less efficient the assembly. Therefore, more advanced students might appreciate the challenge of a more complicated assembly, but less advanced students might want to avoid this. Student materials to explain and design primers for the mutagenesis of GFP are provided in Class Activity 3C: Gibson Assembly Mutagenesis of Fluorescent Protein Genes in Molecular Biology Concepts for Inquiry: The Exploration Workbook. We performed mutagenesis using both the original constitutive promoter BioBridge plasmids and using AraC/pBAD versions of these plasmids created by J. Hackett's students.


Unit 4: The Regulation of Gene Expression

Golden Gate Assembly of Yeast Gene Parts

  • As part of our collaboration with Jef Boeke's lab (Johns Hopkins/NYU) on experiments connected to the Synthetic Yeast Genome project, we created a small library of yeast gene parts, as described on our iGEM wiki. Since the initial iGEM project, we transferred all of our gene parts into kanamycin resistant plasmid backbones to better facilitate Golden Gate assembly. Student materials to explain, design and analyze the assembly of gene parts using Golden Gate are provided in Class Activity 4B: Golden Gate Assembly of Yeast Gene Parts in Molecular Biology Concepts for Inquiry: The Exploration Workbook. If you decide to use this as a lab with your students, I recommend assembling the GAL1 promoter with a GFP coding sequence and the MFA2 terminator in the pAV116 acceptor vector. Then students can observe fluorescence when yeast are grown on 2% galactose, and no fluorescence when yeast are grown on 2% glucose (dextrose). Notes on preparing media are here. Students could also use any yeast promoter in the assembly and then try to observe differences in GFP expression under different conditions, which is what my students have done in the past since we have a promoter library to choose from. While Golden Gate assembly can be performed directly from digested and gel-purified PCR products, I recommend cloning the gene parts into a kanamycin or chloramphenicol resistant vector backbone to allow for ease of assembly into the ampicillin resistant acceptor vector. We perform Golden Gate assemblies according to the BBF RFC 88 protocol developed by Jef Boeke's lab. The calculation of the correct volumes of DNAs to combine in order to create the correct molarities of parts is one of the challenges for students. We transform Golden Gate reactions into DH5alpha or DH10beta bacteria, miniprep plasmid DNA, and then transform the plasmid DNA into yeast. There are multiple yeast transformation protocols such as this one and which you choose will depend, in part, on the timing of having yeast ready to be transformed in class. This lab could start with students designing primers and cloning the gene parts or the instructor could do that and provide part plasmids to the students. Yeast promoters and terminators can be cloned from yeast genomic DNA. Our iGEM lab notebook includes a comparison of safe yeast genomic DNA preps that don't use phenol.

    • Sequences of promoters cloned in our 2012 Dalton iGEM project. You can design primers to add BsaI sequences and sequences for subcloning into a plasmid to the ends of these plasmids.
    • A GFP sequence that has been recoded by the 2012 Johns Hopkins iGEM team to eliminate typeIIS restriction enzyme cut sites: iGEM part BBa_K799028. There are not BsaI site flanking the GFP sequence.
    • MFA2 terminator sequence with BsaI sites deposited as a BioBrick by the 2012 Johns Hopkins iGEM team: iGEM Part:BBa_K799029
    • pAV116, the pRS416 RFP Golden Gate acceptor vector


Induction of RNAi by feeding

  • We induce RNAi by feeding dsRNA-producing E. coli to C. elegans, in a variation of this lab from Cold Spring Harbor DNA Learning Center's silencinggenomes.org.  This lab allows students to observe RNAi and can be used to expose them to the developmental regulation of gene expression as the genes targeted by RNAi are normally active in different cell types and/or different stages of development.  The knockdown of dpy-13 causes the worms to be short and plump. The knockdown of unc-22 causes the worms to have limited movement and to twitch at their ends. The knockdown of bli-1 causes blisters to form due to the separation of a layer of the cuticle from the rest of the worm. dpy-13, and bli-1 are both genes that encode cuticle collagen proteins, but those proteins localize to different parts of the cuticle and their expression peaks at different times, so their knockdown produces different phenotypes.  dpy-13 is expressed throughout larval development, but bli-1 is expressed highly only in the L4 larval stage. Students can be asked to explain this difference in phenotype. The lab can also be used to expose students to how simple model organisms might be used to study human disease. The unc-22 gene is an ortholog of the human gene for Titin and there are multiple genetic diseases associated with this gene, which encodes the largest protein. The lab includes a bioinformatics component including a BLAST search to allow students to find the human ortholog of unc-22. Additionally, this lab can be used to apply concepts of promoter-based regulation of gene expression. The dsRNA is transcribed from two T7 promoters on either side of the RNA-coding sequence and its expression depends on induction of the expression of T7 RNA polymerase from the  lac UV5 promoter using IPTG. The gene for T7 RNA polymerase is integrated in the E. coli genome, while the dsRNA gene for RNAi is on an AmpR plasmid. 
  • There are directions for worm culturing on silencinggenomes.org  and WormBook also provides instructions for culturing C. elegans. The lab calls for an rrf-3 strain of worm because it has better RNAi, but I've found that it works well enough with the N2 worms available from Carolina Biological (Item # 173500). I have a set of OP50 feeder bacteria and RNAi feeder strains (dpy-13, unc-22, bli-1, and empty pL4440 vector or GFP controls) that I've been using, so I haven't tried the kit, but there's also an RNAi kit available from Carolina Biological (Item #211391). These RNAi phenotypes are obvious even in the presence of contaminating wildtype worms. It's also possible to combine this lab with the genetic engineering of the dsRNA-producing plasmids for the feeder strains, but I haven't done that because there's enough other genetic engineering in my course. 
  • Having a cooling incubator so you can keep the worms at 22°C or less and control their growth rate with the temperature is ideal, but just keeping them in a room that's on the cooler side is sufficient. I've had very poor luck with multiple of the lower cost cooling incubators in that they tend to break (stop cooling) after very limited use. If you do purchase one that advertises being able to be used for both cooling and heating, I recommend only using it for cooling and only using it for C. elegans work and you might be able to make it last longer.
  • If you have dissecting scopes with LED lights, that's better for the worms than the hot halogen lights. If your scopes have halogen lights, students shouldn't keep the worm plate on the light for too long at one time.
  • I made my own worm picks by melting broken-off Pasteur pipets onto platinum wire (Platinum Iridium wire, 0.25mm=30Gauge (90%:10%)) using a Bunsen burner.  We use the worm picks to transfer L4 larvae onto the RNAi-feeding plates because these sexually immature worms have not started to reproduce and produce embryos yet. Therefore, all of their progeny will be exposed to the dsRNA throughout their development. Transferring worms with worm picks definitely takes some practice to do well. I recommend that if, as the instructor, you don't have experience with this, that you practice it for a few hours before trying this with students so you can develop a rudimentary enough proficiency to be able to explain the process to students and to be able to finish any of the worm picking that the students aren't able to complete. I draw a picture of the worm pick with its bent, flattened tip on the board and then draw a glob of bacteria on the bottom surface of the tip to represent the "glue" bacteria. Then I tell students that looking in the microscope, they'll need to use the pick to brush the glue over a worm to pick it up. They'll brush in one direction to pick it up and then in the opposite direction to put it down on the surface of the agar near the pool of feeding bacteria on the feeding plate. I ask students to transfer four L4 larvae to each RNAi feeding plate (one RNAi and one control plate per group), and due to scheduling/timing, I've always had to squeeze this into a single 45-minute period. A single period is really not quite long enough, although it's often been easier to make it work in that amount of time when I've had larger classes and a few students have by chance had an easy time (painting experience seems to help) and then can help other groups finish. I emphasize when students are picking worms that it's most important that they not transfer any of the big adult worms. I provide them with multiple images of C. elegans in different stages of development as a reference, but it's still hard for them to be sure if they're seeing L4 worms. I tell them to err on the side of picking younger worms and it doesn't matter if they happen to also transfer a few "baby" worms along with the L4s. They just need to be sure not to transfer adults since each adult could give them 1000 wildtype progeny on their plate. This seems to work well enough for groups to be mostly successful in this lab. Occasionally a group will have lots of wildtype background on their plates - just evidence that they probably transferred adult worms.
  • We observe the RNAi phenotypes on two different days and this is necessary depending on the exact stage of development the transferred worms were in and depending on which gene was knocked down - bli-1 is only evident later in development.
  • My students absolutely love this lab. I think it's completely worth the difficulty of introducing a new model organism, especially because the setup is minimal after the first year of initial setup.

Unit 5: Genome Evolution

Construction of a Phylogenetic Tree


Telomeres and Mutation Rate

  • Students studied the effect of the denaturation of the telomere end-binding protein Cdc13p on the mutation rate was studied as described in using the liquid media and plates described here. These experiments were based on experiments I conducted in graduate school while working with Carol Greider at Johns Hopkins University School of Medicine and used yeast similar to the yeast described in that work. I do not currently have the ability to distribute the wildtype and cdc13-1 loss of heterozygosity strains, but I hope to be able to do so in the future. Please contact me if you would like to be notified if they become available. The genotypes of the yeast are:
    • MATa/MATα cdc13-1/cdc13-1 his3Δ1::pJHU618(GAL1-EST1,HIS3)/HIS3 ade2Δ::hisG/ade2 can1Δ::hphMX/can1 ura3Δ0/ura3 LEU2/leu2 lys2Δ0/LYS2 met15Δ0/MET15 HIS7/his7 TRP1/trp1; VII-L, (ADE5 CAN1 LYS5 CYH2 TRP5 LEU1)/(ade5 lys5 cyh2 trp5 leu1); VII-R, KanMX near the centromere/()
    • MATa/MATα CDC13/CDC13 his3Δ1::pJHU618(GAL1-EST1,HIS3)/HIS3 ade2Δ::hisG/ade2 can1Δ::hphMX/can1 ura3Δ0/ura3 LEU2/leu2 lys2Δ0/LYS2 met15Δ0/MET15 HIS7/his7 TRP1/trp1; VII-L, (ADE5 CAN1 LYS5 CYH2 TRP5 LEU1)/(ade5 lys5 cyh2 trp5 leu1); VII-R, KanMX near the centromere/()


Unit 6: Emerging Molecular Biology, Biotechnology and Medicine

  • I haven't developed any experiments for this unit yet, but it would be a great place to do a lab using CRISPR/Cas9.



Thank you!

Many companies provided free or subsidized reagents or equipment as part of their educational course support, including New England Biolabs, IDT, Gilson, Eppendorf, Promega, and Qiagen.

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