Team:Calgary/Public Engagement/Lesson Plan

Team:Calgary/Public Engagement/Lesson Plan - 2018.igem.org

PUBLIC ENGAGEMENT: LESSON PLAN


Part A: Synthetic Biology 101


Lesson Type: Informal Lecture


Learning Objectives:

  • Define synthetic biology
  • Explain the processes involved in the central dogma of molecular biology
  • Describe the molecular processes behind restriction digestion/ligation, PCR, and gel electrophoresis

Lecture Presentation

  • Ask students if they've ever heard of synthetic biology – do they know what it is?
  • The central dogma of molecular biology is key to understanding and defining synthetic biology
  • The central dogma of molecular biology refers to the transmission of genetic information held in DNA to RNA, then to proteins
  • First, the DNA is used as a template to create RNA by a process called transcription
  • RNA is used as a template to create proteins in a process called translation
  • DNA is a double-stranded molecule twisted in the shape of a double-helix
  • The information in DNA, also known as the "genetic code", is stored in the sequence of bases (adenine, guanine, cytosine, and thymine)
  • Adenine pairs with thymine, while cytosine pairs with guanine. Each line in the DNA diagram represents one of these "pairs"
  • Every individual has unique DNA code
  • DNA is converted to RNA by the process of transcription
  • RNA is a single-stranded molecule
  • RNA sequence is derived from the sequence of base pairs on a DNA molecule
  • Instead of thymine, RNA contains a uracil base
  • Not all DNA is converted to RNA: only certain parts of DNA that can code for proteins are converted to RNA
  • RNA is converted to proteins through the process of translation
  • Every three bases in RNA codes for a single amino acid. For example, AUG codes for methionine
  • There are only four bases, but 20 different amino acids
  • Proteins have many functions within a cell
  • They can synthesize molecules, breakdown molecules
  • Synthetic biology can be defined as the laboratory editing of any of the processes involved in the central dogma
  • DNA, RNA, or proteins can be edited to make the cell do different things
  • Can manipulate the colour, shape, size, function, etc. of the cells
  • For example, if you put a gene that codes for the red fluorescent protein (RFP) in E. coli, the cell will glow red
  • There are many ways in which synthetic biologists can edit DNA, RNA, or Proteins
  • There are three common techniques that are very important for the work that scientists do in the lab
    • Digestion/ligation
    • PCR amplification
    • Gel electrophoresis
  • Digestions/ligations are a way to insert new fragments of DNA into an existing piece of DNA
  • Special proteins called "restriction enzymes" can recognize specific sequences called "restriction sites" in the DNA
  • The restriction enzyme will bind to the DNA and cut the DNA at that specific sequence
  • The process of the DNA being cut by a restriction enzyme is called a "restriction digestion"
  • Restriction enzymes often cut the DNA twice: once on each strand
  • They often cut close together but not on the exact same spot, resulting in "DNA overhangs", which are short sequences of single-stranded DNA
  • The DNA insert is designed to have ends with overhangs that match the overhangs on our digested DNA
  • The overhangs match with one another and the DNA comes together
  • DNA ligase is used to seal the DNA together
  • Repaired DNA is now stable and can be transcribed and translated
  • The next technique we will look at is PCR amplification
  • PCR, which stands for "polymerase chain reaction", is a method of amplifying a small section of DNA
  • This method works by first denaturing (separating strands) of DNA by heating it to 95 degrees
  • Lowering the temperature attaches "primers" onto the DNA
  • Primers are short DNA sequences that flank the region you want to amplify
  • Afterwards, DNA is heated to 75 degrees to synthesize DNA
  • The PCR reaction repeats these steps for 30-40 cycles
  • PCR amplifies the fragment of DNA between the two primers exponentially
  • This is because after each cycle, the newly synthesized DNA can act as a "template" DNA for the next cycle
  • Gel electrophoresis is used to identify the lengths of DNA fragments
  • DNA is loaded into wells at the top of a gel and a current is run through
  • DNA, being negatively charged, will travel towards through pores in the gel to the positive end of the gel
  • Longer strands of DNA will face more friction from the pores, and travel slower than shorter bands, which will travel further down the gel
  • The DNA separates into "bands" in accordance to their size
  • When visualized under UV light, the bands fluoresce
  • The bands can be compared with a DNA "ladder" of known lengths to determine their size
  • Enjoy the Strawberry DNA Extraction activity!

Part B: Strawberry DNA Extraction


Lesson Type: Demonstration/Performance


Learning Objectives:

  • Describe how DNA is packaged within the cell
  • Explain the differences between strawberry cells and human cells
  • Understand how histones are removed from DNA

Background

The adult human body contains trillions of cells - each incredibly small and impossible to see with the naked eye. Within each of those cells, there is a bundle of DNA called the nucleus which contains almost all of your genetic information. Inside each nucleus, which is 6 micrometers in diameter, are 46 chromosomes of DNA, for a total of 3 billion base pairs. Although each base pair is microscopically small, we have enough DNA in each of our cells to amount to 2 meters in length when stretched out. Inside our cells, our DNA doesn’t exist as elongated strands. Instead, the DNA in our cell is wrapped tightly around proteins called histones, allowing for large stretches of DNA to be condensed into a structure called chromatin.

Humans are a diploid organism, meaning we have two copies of each of the 23 chromosomes in our cells. Strawberries, which we will be extracting DNA from today, has 8 copies of each chromosome, making it an octoploid organism. Because they have so many copies of each chromosome, its cells contain much more DNA than our cells do, making the DNA easier to see when removed from the cell.

DNA in strawberry cells can be made visible to the naked eye as long as many cells are broken open and the DNA is separated from the histones that package the DNA. First, the cells are broken apart using a detergent that disrupts the membrane layer of the cell. Then, DNA is separated from the histones via simple ionic interactions. When the cell is flooded with aqueous NaCl, the Na+ ions bind to the negatively-charged DNA molecules, and the Cl- ions bind to the positively-charged histones, separating the two. This mixture is filtered to remove excess cell debris. Lastly, a layer of alcohol is added to the mixture. DNA, which is soluble in water and insoluble in alcohol, will collect at the meeting between the water and alcohol layers.


Materials (per student):

  • 2 strawberries
  • 1 ziploc bag
  • 20mL strawberry extraction buffer
    • 5 reactions of buffer can be made from
      • 10mL detergent
      • 90mL water
      • 1.5g of NaCl
  • 1 coffee filter
  • 1 beaker or plastic cup
  • 10mL ethanol
  • 1 stir rod or toothpick

Procedure:

  1. Place a strawberry in a ziploc bag. Seal the bag and squish the strawberry in the bag until the mixture is smooth.
  2. Measure 20 mL of the strawberry extraction buffer and pour it into the ziploc bag.
  3. Seal the bag and gently mix the contents. Try to avoid forming bubbles in the bag.
  4. Place a coffee filter inside a beaker or plastic cup. Pour the contents of the bag into the coffee filter. Let sit for 10 minutes.
  5. Gently squeeze the sides of the filter paper to push any remaining liquid through the filter paper.
  6. Measure 10 mL of ethanol and pour gently down the side of the cup. Let sit for 5 minutes.
  7. Use a toothpick or stir rod to swirl in between the alcohol and water layers. Remove toothpick to see DNA!

Part C: Evaluation of Understanding


Lesson Type: Worksheet and/or Quiz


This section may be modified based at the discretion of the teacher. These sample questions can be used as a part of a stand-alone quiz for this lecture, or integrated as a part of a unit test.


Multiple Choice Questions

1. Restriction Enzymes _____ the DNA, while DNA ligases _____ the DNA.

  1. Fix, Cut
  2. Repair, Mutate
  3. Mutate, Repair
  4. Cut, Fix

2. PCR stands for _____.

  1. Polymerase Chain Reaction
  2. Protein Coding Reaction
  3. Polymerase Chain Repair
  4. Protein Coding Reaction

3. In gel electrophoresis, shorter DNA fragments appear at the ____ of the gel, while longer DNA fragments appear at the ____.

  1. Top, Bottom
  2. Left, Right
  3. Right, Left
  4. Bottom, Top

4. The Central Dogma of Molecular Biology is the process in which information flows from ____ to ____ to ____.

  1. RNA, DNA, Protein
  2. Protein, RNA, DNA
  3. DNA, RNA, Protein
  4. DNA, Protein, RNA

5. DNA is _____, while RNA is _____.

  1. Double-stranded, single-stranded
  2. Double-helix, single-helix
  3. Double-stranded, single-helix
  4. Double-helix, single-stranded

Short Answer Questions

1. How is DNA packaged into a small cell?

2. What does the addition of NaCl do to the DNA in lysed strawberry cells?

3. Explain the steps involved in 1 cycle of the PCR Reaction.


Answers

Multiple Choice

1. D; 2. A; 3. D; 4. C; 5. A

Short Answer

1. DNA is "packaged" by tightly winding around proteins called histones to create a structure called chromatin.

2. Positively charged Na+ ions bind to negatively charged DNA, and negatively charged Cl- binds to positively-charged histones, resulting in the separation of DNA from histones.

3. The DNA strand is denatured (separated into two strands) at 95 degrees. At 55 degrees, the PCR primers anneal to the DNA. afterwards, the DNA is heated to 72 degrees, stimulating Taq DNA polymerase activity, resulting in DNA elongation.


Download the printable lesson plan here.


Download the powerpoint here.