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BioBits

TM. @ biobitsproject @ miniPCR. f b.com/ biobitsproject fb.com/ miniPCR. BioBits. Cutting-edge synthetic biology in your hands. @ biobitsproject @ miniPCR. The Central Dogma of Molecular Biology.

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BioBits

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  1. TM @biobitsproject @miniPCR fb.com/biobitsproject fb.com/miniPCR • BioBits • Cutting-edge synthetic biology in your hands • @biobitsproject • @miniPCR

  2. The Central Dogma of Molecular Biology The central dogma is a guiding principle for how information can be transferred between DNA, RNA, and protein It states that information can be passed between DNA and RNA and into protein, but information cannot be passed from protein to DNA or RNA

  3. The Central Dogma of Molecular Biology Often, the central dogma is simplified to represent the most common flow of information, from DNA to RNA to protein We usually think of this occurring in a two step process: DNA to RNA in a process called transcription RNA to protein in a process called translation TM

  4. Transcription What molecules or cellular components are needed for transcription? • RNA polymerase • Ribonucleotides • ATP TM

  5. Translation What molecules or cellular components are needed for translation? • Ribosomes • tRNAs • Amino acids • ATP TM

  6. BioBits™ can do this without cells Freeze-dried, cell-free • Cell-free: contains the extracted cellular tools but no live cells • Freeze-dried: can ship and store at room temperature • What else do we need to add to BioBits™ pellets for transcription and translation to happen? • DNA + water TM

  7. The DNA for today’s lab In this lab, the DNA that we will add to our tubes contains a gene that allows us to observe transcription and translation in real-time Aptamer: a sequence in the mRNA that will fold and bind to a green fluorophore present in the BioBits™ pellet When mRNA is transcribed from DNA, the solution will fluoresce green TM

  8. The DNA for today’s lab In this lab, the DNA that we will add to our tubes contains a gene that allows us to observe transcription and translation in real time Red fluorescent protein: the protein coded for by this sequence will fluoresce red when exposed to blue light When the mRNA is translated to protein the solution will fluoresce red TM

  9. Making transcription and translation visible DNA is transparent in solution Aptamer for green fluorescence Red fluorescent protein TM

  10. Pre-labTube 1: BioBits™ pellet + water What do we call tube 1? A negative control What do we learn from including tube 1 in this experiment? A control gives us something to compare our other samples to Without the control, we don’t know if just adding water alone is the reason for our observations TM

  11. Pre-labTube 2: BioBits™ pellet + DNA A Tube 2 contains everything needed for transcription and translation to occur TM

  12. Pre-labTube 3: BioBits™ pellet + DNA A + Kanamycin Kanamycin is a drug that binds to prokaryotic ribosomes like those found in BioBits™ pellets It blocks the mRNA and tRNAs from aligning properly This leads to significant errors in during translation TM

  13. Pre-labTube 4: BioBits™ pellet + DNA B For tube 4, your job is to observe the reaction Based on your observations you must deduce what was different about this reaction ? ? ? TM

  14. Pre-labPredictions Justify your predictions ? ? ? TM

  15. Open BioBits™ carefully! Tap BioBits™ strip tubes gently on the counter to collect the freeze-dried pellets at the bottom of the tube Carefully remove each cap in the strip, one at a time, taking care not to dislodge the BioBits™ pellet TM

  16. Let’s get started! Add reagents to your tubes Observe your reactions in P51™ fluorescence viewer or other blue light illuminator Record your initial observations in Table 2 of the Student’s Guide TM

  17. Protocol: Incubate 15 minutes Incubate your tubes at 37°C for 15 minutes Use a miniPCR thermal cycler in heat block mode, a water bath or incubator You may also use body heat: hold the tubes in a clenched fist or other place close to the body such as a pocket or under your arm After 15 minutes, observe your reactions in P51™ fluorescence viewer or other blue light illuminator Record your observations in Table 2 of the Student’s Guide TM

  18. Protocol: Now wait at least 8 hours Your final observation can be made between 8 and 72 hours after starting the reactions (typically, 24 h) Incubate your tubes at room temperature over this time TM

  19. Day 2 @biobitsproject @miniPCR fb.com/biobitsproject fb.com/miniPCR • What will we see? • @biobitsproject • @miniPCR

  20. Protocol Observe your reactions in P51™ fluorescence viewer or other blue light illuminator Record your observations in Table 2 of the Student’s Guide TM

  21. Results: Tube 1 What did you observe at • Time 0 • 15 min • Day 2? Did this match your predictions? Explain your observations Red fluorescent protein Aptamer for green fluorescence DNA is transparent in solution TM

  22. Results: Tube 2 What did you observe at • Time 0 • 15 min • Day 2? Did this match your predictions? Explain your observations Red fluorescent protein Aptamer for green fluorescence DNA is transparent in solution TM

  23. Results: Tube 3 What did you observe at • Time 0 • 15 min • Day 2? Did this match your predictions? Explain your observations Red fluorescent protein Aptamer for green fluorescence DNA is transparent in solution TM

  24. Results: Tube 4 What did you observe at • Time 0 • 15 min • Day 2? Did this match your predictions? Explain your observations Red fluorescent protein Aptamer for green fluorescence DNA is transparent in solution TM

  25. Discussion How did this experiment demonstrate the central dogma? What are other ways in which this process can be interrupted? What are the advantages of doing this lab in a cell-free system? Disadvantages? TM

  26. TM

  27. Extension: RNA aptamers Which strand of the DNA is the template? Mark with a 3’ and 5’. Find the Broccoli units in the sequence. Find the midpoint of the stem loop structure. Why is the sequence/structure not symmetrical? 3’ 5’ A B B A TM

  28. Extension: cell-free technology Read about the development of BioBits™ Read more about how cell-free technology works and how scientists are using it Huang, Ally. “BioBits™ Explorer: A modular synthetic biology education kit.” Link. Stark, Jessica. “BioBits™ Bright: A fluorescent synthetic biology education kit.” Link. Stark, Jessica. “BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts” Link. TM

  29. Extra Slides

  30. Pre-lab Protocol: overview Add reagents to your tubes Observe your reactions in P51™ fluorescence viewer or other blue light illuminator Incubate at 37˚C (incubator or body heat) for 15 minutes Observe your reactions in P51™ fluorescence viewer or other blue light illuminator Leave tubes at room temperature overnight On Day 2, observe reactions in P51™ fluorescence viewer or other blue light illuminator TM

  31. Pre-lab

  32. P51™ Learning Labs How did this experiment demonstrate the central dogma? What are other ways that this process can be interrupted? What are the advantages of doing this lab in a cell-free system? Disadvantages? TM

  33. P51™ Learning Labs How did this experiment demonstrate the central dogma? What are other ways that this process can be interrupted? What are the advantages of doing this lab in a cell-free system? Disadvantages? TM

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