Team:Marburg/Communication/BioBits

BioBits

"Tell Me and I Will Forget; Show Me and I May Remember; Involve Me and I Will Understand." - Confucius

In order to conduct a hands-on experiment with the students of two biology courses, we resorted to the BioBits kits from the company miniPCR. This offering was not only very exciting and entertaining for the students, but also served as a pilot project for long-term implementation as part of our extensive educational work at iGEM.

BioBits is an excellent way to present the topic of the central dogma of molecular biology. Students learn both basics and more advanced details about DNA, RNA, and the process of protein synthesis in a two-day experiment.

The first day begins with a theoretical introduction to the topic. Here the presentation provided by miniPCR can be used, which we translated to German for the students. After establishing a sufficient basis of understanding for protein synthesis, the instructions for experiment execution and description began. A course we created on the correct handling of the pipettes was also integrated into the presentation.

In the experiment, both transcription and translation are visualized separately from each other by fluorescence. The students are presented with four tubes for this purpose. Each of the tubes has a pellet containing the necessary components of a freeze-dried Cell-Free System from E.Coli. These include polymerases, ribosomes, and other cellular components for building proteins. As additional reagents, students will receive four aliquots that our team prepared from the original vessels for the individual groups, as recommended in the miniPCR instructions.
The four reagents are water (nucleasease free), DNA A, DNA B, and kanamycin, an antibiotic that inhibits the S30 subunit of bacterial ribosomes, thereby preventing translation. In addition, we provided micropipettes with tips, as well as P51™ Molecular Fluorescence Viewers.

The structure of DNA A is explained to the students from the beginning. Here, it is encoded twice for a green fluorescent aptamer, an RNA structure, as well as a red fluorescent protein. DNA B does not contain any given information, the students are to make their own hypotheses for the content of the DNA structure at the end of the experiment.

First, the tubes containing the pellets were illuminated without liquid in the Fluorescence Viewer. Due to the refraction of the light, the tubes fluoresced slightly greenish even without contents. As soon as water is added, the refractive index changes and the fluorescence stops. When asked how we could ensure in the experiment that the tubes with liquid would not fluoresce green even without active aptamers, the students correctly determined that a negative sample was necessary.

Therefore, in the four tubes, a negative sample was filled with water only, a sample of DNA A and one of DNA B were taken, and a tube in which DNA A was mixed together with kanamycin. The students were asked to guess what results could be expected from the DNA A samples when they were prepared, after 15 minutes and after two days. No fluorescence can be seen at the start of the test series. After 15 minutes, green fluorescence should be visible in the P51™ Molecular Fluorescence Viewer due to expression of the RNA with the formation of the aptamers in contrast to the negative control. A red glow is not expected to occur until the second day, as translation takes longer than transcription. However, in the tube containing DNA A and kanamycin, translation should not occur and thus red fluorescence should be absent. The aptamers will decay by day two since they are made of unstable RNA.

Most of the student groups found themselves very comfortable with the instructions given and the pipettes provided. In groups of two to three, each student was able to actively participate. After pipetting the aliquots into the tubes, the students warmed them in their hands to speed up the transcription. At this point, we took the opportunity to include a brief explanation of enzyme kinetics.

After 15 minutes of incubation, students viewed their tubes in the P51™ Molecular Fluorescence Viewers. For most, as expected, the negative control was dark, DNA A with water or kanamycin was equally green, and DNA B also fluoresced greenish.

We collected the tubes and stored them in a cabinet protected from light until the second day. Here, the students started directly observing their results. As shown in the picture, only the tube containing DNA A and water had turned red. The green fluorescence of the aptamers had decreased as expected.

Now it was up to the students to draw conclusions from the collected observations. All tubes with DNA showed clear signs of transcription by expression of the aptamers, and this result could be confirmed by comparison with the negative control. However, red fluorescence as a sign of translation occurred in only one tube, and the absence was expected for the kanamycin tube because the antibiotic inhibits translation. DNA B, however, remained as an open question.

Students made various guesses as to why DNA B did not contain red protein despite the fact that translation was actually possible, since RNA was transcribed and no kanamycin was added. Since the structure of the protein is instrumental in its ability for fluorescence, some students posited various types of mutations that could have caused the protein to have a different structure compared to DNA A. The resolution was that DNA B did not contain red protein despite the fact that RNA was transcribed and kanamycin was added.

In this experiment, students were able to engage with the concepts of molecular biology in a multi-faceted way. Protein synthesis with its individual steps could be vividly experienced and conclusions drawn from the methodology to an unknown DNA sequence.

We as a team are extremely satisfied with the course of the event. The students and the teacher of the two courses present were also extremely positive. In order to bring this experience to schools in Germany after our iGEM project, we were able to cooperate with The German Association for Synthetic Biology (GASB).