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Frankenfood or Golden Rice

Work with a genetically modified organism that can be used to bake breads containing a super-dose of Vitamin A

INTRODUCTION

Genetically-modified foods are sometimes described as life-saving approaches to malnutrition, and sometimes protested as unnatural and unsafe. In this series of labs, you will work with a genetically modified organism that can be used to bake breads containing a super-dose of Vitamin A. You will test the bright orange yeast for vitamin production and for the food quality. Classroom time will be used to understand the genetic changes inside these “VitaYeast,” as well as to debate the merits/pitfalls of food re-engineering.

LEARN

  • Sterile microbial techniques to grow yeast in the laboratory
  • To perform quality control tests on genetically altered yeast cells
  • To investigate the genetics of vitamin production and how to engineer it
  • To debate the ethics of tinkering with our food supply

WORKFLOW FOR THIS EXPERIMENT

SampleWorkflow_VitaYeast.pdf
Adobe Acrobat document [215.4 KB]

Procedure

Before you begin your work you should put on personal protective equipment as deemed appropriate by your school. These may include

  • Nitrile gloves,
  • Eye protection
  • lab coat

GETTING READY

TECHNIQUES TO LEARN

In advance of the transformation lab will need to learn sterile technique (shown in “liquid ONs” video), how to use the pipets and how to spread the bacteria on the plates. Videos for these three basic techniques are shown below.

 

GROWING VITA-YEAST

POURING DOUGH MEDIA

  1. Place a weigh boat on a top loading balance and tare the balance.
  2. Measure 2g of NaCl, using a plastic spoon to remove the salt from the stock container.
  3. Add the salt to the 100 ml bottle, and discard the weigh boat.
  4. Place a new weigh boat on the balance, tare it, and then weigh 3g of All-Purpose Flour.
  5. Add the flour to the 100 ml bottle that has the salt, and discard the weigh boat.
  6. Place a new weigh boat on the balance, tare it, and then weigh 1g of Agar or Gelatin.
  7. Add the Agar to the 100 ml bottle that has the salt and the flour, and discard the weigh boat.
  8. Measure 50 ml of water with the 50 ml conical tube and add it to the 100 ml bottle. Q: how does this recipe compare to the recipe for baking bread?
  9. Swirl the mixture for 10 or 15 seconds to try to mix the water into the powders as best you can.
  10. Screw on the cap to the 100 ml bottle VERY LOOSELY. If the cap is on too tight when you microwave the mixture, the pressure that builds up inside the bottle may break it.
  11. CAREFULLY: microwave the mixture for 20 seconds at a time, using a hot mitt to hold the bottle and swirl the mixture. It will remain cloudy and very likely lumpy.
  12. Microwave until the liquid that swirls around seems thick and hot.
  13. Pour the liquid into a petri dish or two, depending on how much was dissolved. Pour only until the bottom of the petri dish is covered. If you pour some lumps into the media, that’s OK.
  14. Cover the bottom section of the petri dish with the top and allow the media to solidify undisturbed on the benchtop, 30 minutes or so.

RESTREAKING YEAST

  1. Label your new petri dish with your initials, today’s date, the kind of media in the petri dish and the strain that you’ll be restreaking onto it.

    Restreaking Yeast for Single Colonies

  2. Start by dabbing the flat end of a toothpick onto a colony of yeast or bacteria that you want to restreak. The colony should be well isolated from the others and uniform in appearance.
  3. Transfer the cells from that toothpick by lightlying touch the toothpick to a spot on the new petri dish that you’d like to grow. Note: you should not break the surface of the agar with any of this procedure, but the results will still be OK, even if you do.
  4. With the flat end of a new toothpick, spread out the cells in the dab you made on the new petri dish by drawing your toothpick back and forth through the dab and then along the media in the dish. Do not back up as you draw since you are trying to spread out the cells that are on the toothpick from your one pass through the original dab of cells.
  5. With a new toothpick, spread out the cells still further, drawing from the ending line you made with the second toothpick. Again, do not back up as you draw with this third toothpick and try not to break the surface of the media.
  6. Replace the lid of the petri dish and incubate the plate media-side UP in an incubator (ideally 37° overnight for bacteria, 30° 2 days for yeast).

LIQUID OVERNIGHT CULTURES

VOCABULARY TO LEARN:

“inoculate” = add a small amount of a material to get a starting reaction going.

“aliquot” = to doll out a portion of a material, sort of like a serving size.

  1. Examine the bottle of YPD to confirm that it is clear and nothing fuzzy or cloudy is growing in it.
  2. Label the two tubes with your initials, today’s date, and either “+cells” or “no cells.”
  3. Using sterile technique, place a 5 ml serological pipet in a pipet bulb or aid.
  4. Withdraw 5 ml of YPD into the pipet.
  5. Aliquot 2.5ml of media to each tube, replacing the lid of the tubes quickly.
  6. Dispose of the pipet in a waste container for sharps.
  7. Use a toothpick to scoop a colony of cells from the petri dish and throw the colony into the media in the tube marked “+Cells.”
  8. Replace the cap and grow the cultures overnight at room temperature or 30° on a rocker platform or roller wheel.

THIN LAYER CHROMATOGRAPHY

MAKING TLC SLIDES

VOCABULARY TO LEARN:

“tare” = to zero a balance, in other words to remove the weight of the container from the measurement

  1. Set out two microscope slides onto a paper towel.
  2. Use a sharpie to label the slides with your initials and today’s date.
  3. Turn the slides over so the ink is facing down.
  4. Place a weigh boat on a top loading balance and tare the balance.
  5. Measure 1 g of Plaster of Paris using a plastic spoon to remove the powder from the stock container.
  6. In a new weigh boat, measure 1 g of Cornstarch or powder.
  7. Mix the Plaster of Paris with the Cornstarch in a single weigh boat.
  8. Using a transfer pipet, add 3 ml of water to the powders.
  9. Quickly mix the water into the powders to form a smooth slurry. You can use the transfer pipet as a mixing rod or to pipet the slurry up and down.
  10. Use the transfer pipet to move the slurry from weigh boat to the surface of the microscope slide you prepared, using the transfer pipet to smooth the surface and remove bubbles.
  11. Allow the slides to sit on the bench until they have dried, approximately 30’-one hour.

VITAMIN A STOCK

For experiments that need a stock of Vitamin A, you will have to snip the end of a capsule off, and squeeze the oily liquid into an eppendorf tube. There will likely be around 100 ul of solution that you can then pipet as needed. Be sure to label your eppendorf tube to note the contents of the tube and the date.


YEAST EXTRACTION

  1. Mix the starting liquid overnight culture so it’s homogeneous. This can be done by swirling the culture if it’s in a flask, by inverting if the cap is tight, by flicking a small tube, or by vortexing if there is a vortex available.
  2. Place a tip on your pipetman and move 500 ul of the culture to an eppendorf tube.
  3. Using an eppendorf tube with end ½ trimmed off at the 100 ul mark, measure 100 ul of small glass beads and add them to the 500 ul of yeast you measured out.
  4. Close the eppendorf tube.
  5. Vortex the mixture of yeast and glass beads, keeping the tube on the vortex at full speed for 15 seconds and then letting the solution “rest” for 15 seconds. This rest is needed to keep the temperature of the yeast/beads mixture from overheating.
  6. Repeat the vortexing and resting series a total of 4 times so the yeast will have been mixed with the beads for a full minute.
  7. Allow the beads to sink to the bottom of the solution and then remove the solution of yeast extract from the top, using a pipetman and tip and a new eppendorf tube.
  8. Label the eppendorf tube that has your yeast extract with its contents, your initials and today’s date.

THIN LAYER CHROMATOGRAPHY

  1. Begin by putting on gloves to protect the TLC sheets from any oils or pigments on your hands
  2. Use a microscope slide to measure and a pencil to mark on the white side of the TLC sheet the size of the piece you’ll need to cut.
  3. Cut the sheet to size.
  4. Mark the starting line for your materials by measuring 1.5 cm from one of the narrow ends and lightly drawing a line across the white surface of the TLC slide. Do not dig deeply into the material on the surface.
  5. Spot 3 ul of Vitamin A about 1/3 of the way across the line you drew at the bottom of the slide.
  6. Spot 3 ul of Yeast extract about 2/3 of the way across the line you drew at the bottom of the slide.
  7. Allow the spots to dry while you make up the solvent that you’ll use to carry the vitamins up the TLC slide. Mix 9 ml of water with 500 ul EtOH and 500 ul Isopropanol. These can be mixed directly in a 50 ml conical tube. Label the tube with the contents, your initials and today’s date.
  8. Lean the TLC slide in the 50 ml conical tube, keeping the upper edge of the solvent below the line where you’ve spotted the vitamins. The idea is that the solvent must pass through the vitamins to carry them along as the solvent gets wicked up the slide.
  9. Replace the lid on the 50 ml conical tube and allow the TLC to proceed undisturbed for 20 or 30 minutes.
  10. Using a gloved hand, remove the slide from the conical tube.
  11. Allow the solvent to evaporate and then visualize the extent and the intensity of the materials that have moved along the slide, using a UV lamp to visualize the vitamin.

BAKING BREAD

DISSOLVING YEAST

VOCABULARY TO LEARN:

“YPD” = rich yeast growth media, like chicken soup for yeast.

  1. Check the expiration date of baker’s yeast and make a note of the date in your lab notebook.
  2. Label the flask with labeling tape and write “baker’s yeast” and your initials and today’s date on the label.
  3. Open the package of baker’s yeast and pour the contents into the sterile flask.
  4. Examine the bottle of YPD to confirm that it is clear and nothing fuzzy or cloudy is growing in it.
  5. Using the conical tube and sterile technique, measure 50 ml of YPD and pour the media into the flask.
  6. Repeat so the volume of YPD in the flask is 100 ml.
  7. Replace the cover of the flask.
  8. Place on the stir plate and set to stir gently until yeast is dissolved.
  9. Make notes on appearance of yeast solution at time = 0’, time = 1’, time = 5’, time = 15’

SUBCULTURING YEAST

VOCABULARY TO LEARN:

“subculture” = to dilute a densely grown population of cells into fresh media to allow for active growth and to produce more cells.

  1. Label the flask with labeling tape and write “VitaYeast” and your initials and today’s date on the label.
  2. Mix the starting liquid overnight culture so it’s homogeneous. This can be done by inverting if the cap is tight, or by flicking or by vortexing if there is a vortex available.
  3. Examine the bottle of YPD to confirm that it is clear and nothing fuzzy or cloudy is growing in it.
  4. Using the conical tube and sterile technique, measure 50 ml of YPD and pour the media into the flask.
  5. Repeat so the volume of YPD in the flask is 100 ml.
  6. Using sterile technique, remove 1 ml of the overnight culture of yeast using your 5 ml serological pipet attached to a pipet bulb or pipet aid.
  7. Transfer the yeast into the 100 ml of YPD media in the flask.
  8. Dispose of the serological pipet in bio-hazardous or sharps waste.
  9. Replace the cover of the flask.
  10. Place on the stir plate and set to stir gently overnight.
  11. If there is time you may want to measure the starting density of the culture using 1 ml in a cuvette and a spectrophotometer set to 600 nm. Blank the spectrophotometer with YPD.

SERIAL DILUTIONS

  1. To prepare a series of 1:10 dilutions, you should add 900 ul of water to the cuvettes you’ll be using. If you are making these dilutions in glass tubes, you may need 2.7 ml of water. These volumes can be measured with a serological pipet and a bulb.
  2. Make sure that your starting liquid culture or solution is homogenous. You can mix by inverting if the cap is tight, or by flicking or by vortexing if there is a vortex available.
  3. Place a tip on the end of your pipetman and then remove 100 ul of the culture or solution and place it in the first tube with 900 ul of water (or remove 300 ul of culture if diluting to 2.7 ml water).
  4. Dispose of the pipet tip into bio-hazardous or sharps waste.
  5. Label that dilution “1:10” and then invert or gently vortex to mix completely.
  6. Repeat steps 3, 4 and 5, moving the 1:10 dilution to a fresh tube or cuvette with water to make a 1:100 dilution.
  7. Repeat using the 1:100 dilution to make a 1:1000 dilution.
  8. Using a tube or cuvette with water to blank the spectrophotometer, measure the optical density or absorbance of each dilution. Q: do you expect each to be precisely 1/10th of the last? Why or why not?

You may be asked to follow up this experiment with some statistics and analysis of your data.