CMB Education » Biomedical Technology » CellbiologyPractical

KTH Cell and molecular Biology

Biomedical technology

Cell biology practical

Contents

  • The practical

  • Lab safety

  • Practical: Fluorescent proteins as reporters

  • Day 0: Introduction lecture with laboratory safety
  • Day 1: Transformation of E. coli with DNA
  • Day 2: Results of transformation, restreaking on new plates. Discussion on biomedical self-testing.
  • Day 3: Results, analysis and discussion. Introduction to microscopy.
  • Day 4: Reserve day (in case needed)

The practical

  • The aim of the practical is to introduce the students to some basic methods in cell biology
    • Knowledge of basic laboratory safety
    • The basics of sterile work with living organisms
    • Understanding of the process of genetic transformation
    • The basics of the use of fluorescent reporter molecules in cell biology
    • The basics of microscopy (light microscopes, fluorescence microscopes)
    • The basics of recombinant protein expression

  • Participation is not obligatory (similar status as lectures)
  • The students MUST be there every day when the exercise begins
  • Students coming in late and missing the roll call will not be let into the laboratory that day
  • The practical is done in small groups of 2-3 people
  • The contents will be examined in the final exam in the end of the course
  • No laboratory report required

Laboratory safety

  • Come prepared to the laboratory – make sure you read the compendium before the laboratory.
  • There is always time to think first before you do anything.
  • If you don’t know how to do a specific procedure, read the instructions or ask an instruction. Don’t guess.
  • There are trained instructors available in the course laboratory at times.
  • If you then still don’t know how to do something, don’t.

In the laboratory

  • You must wear a lab coat. On this course, lab coats are provided to you. Leave them where you found them.
  • You may not take your bags into the laboratory.
  • Eating and drinking are strictly forbidden in the laboratory.
  • Find out where emergency showers and first aid gear are located in the laboratory.
  • Find out where emergency exits are.
  • Organize your work place with all the materials needed before you start pipetting or whatever.
  • Keep an eye on your neighbor. Safety is a joint venture.
  • Wear gloves of the right size when you work. Gloves are disposable and cheap.
  • Protect your eyes, face and arms when working with ultraviolet light.
  • All contaminated waste is must be placed in the special yellow containers in the laboratory.
  • Wash your hands when leaving the laboratory.
  • Hands have not been washed properly unless they have been washed with soap.

Practical: Introduction

One of the methods in modern cell biology is genetic modification of organisms, where the genetic makeup of an organism is altered. In this practical, we will transform a laboratory strain of E. coli with genes isolated from a species of jellyfish. These genes code for special fluorescent proteins (such as GFP, green fluorescent protein) that the jellyfish uses for communicating or luring prey.

The word “transformation” dates back to the 1940’s when scientists showed strains of bacteria could be “transformed” by some sort of hereditary material. Later, this material was shown to be DNA. Today, transformation is a common laboratory procedure. Plasmids are small circular pieces of DNA, which are used to transform E. coli. Because they are circular, like bacterial chromosomes, they replicate when the cells divide, and are distributed into the daughter cells. The plasmids can be isolated and purified from the bacterial cells, and modified in various ways.

There are many ways of getting E. coli to accept foreign DNA from its environment and thus become transformed. The most common and simplest is by a heat shock in a calcium chloride solution. The heat shock carries the DNA into the cell. This process is not very efficient and only a small fraction of the bacterial cells actually receive the DNA. To identify the successfully transformed bacterial cells, a selectable marker gene is used on the plasmic. Typically the marker gene confers resistance to an antibiotic. The transformed bacteria containing the plasmid with the antibiotic resistance gene are able to survive on a petri dish containing growth medium with the antibiotic. The bacteria that did not receive the plasmid will not grow on these plates. The three plasmids used in this practical contain an ampicillin resistance gene (as selection marker) and genes for different fluorescent proteins. The bacteria carrying these plasmids will express the fluorescent proteins that can be visualized in a fluorescence microscope.

For more information on fluorescent proteins and fluorescence microscopy, study the following links:

Practical day 0: Introduction

  • Introductory lecture with safety aspects
  • Read the whole manual and try to simulate the steps in your mind

Practical day 1: Transformation of E. coli

Materials:

  • overnight culture E. coli laboratory strain DH5 grown a plate without antibiotic
  • three empty, sterile Eppendorf tubes
  • three 1,5 ml Eppendorf tubes containing the three plasmids
    • pDsRed2
    • pECFP
    • pGFPuv
  • sterile 100 mM calcium chloride solution on ice
  • ice bucket or any small container with some ice (can share with neighbors)
  • sterile Eppendorf tubes (1,5 ml)
  • Gilson pipettes and sterile tips to Gilson pipettes
  • a water bath with water at +42 Centigrades, a raft for
  • an incubator for plates at +37 Centigrade
  • sterile glass beads for spreading the bacteria on plates
  • three plates (Petri dishes) with rich medium and ampicillin (antibiotic), marked with red (Amp)
  • disposable gloves

Procedure

  1. Make an inventory. Set up your “station” on the bench.
  2. Find the +42 Centigrade water bath and the +37 Centigrade incubator.
  3. Use a glove and a piece of tape to make your own disposable wastebasket on the bench.
  4. Wear gloves all the time. Do not touch the inside of the cap with your gloves since the gloves are not sterile.
  5. Set up three separate transformations experiments (you have three different plasmids to use) with the three empty sterile Eppendorf tubes. Mark the tubes on the lid. Place the tubes on a small volume of ice.
  6. Using a sterile tip on a Gilson, add 200 microliters of the 100 mM calcium chloride solution to each of the three 1,5 ml Eppendorf tubes.
  7. With a new sterile Gilson tip, remove a tiny, tiny little bit of bacterial mass from the plate (like the size of an o on this page) and add it to each one of the three tubes. Mix carefully. If you pipette the mix up and down in your Gilson, make sure that you don’t get any of the mix inside of the pipette.
  8. Keep the three tubes with E. coli on ice and wait 15 minutes (approximately). Note the starting time.
  9. During these fifteen minutes, get three plates supplemented with ampicillin. Mark the plates on the bottom with your names, the date and the plasmid, to identify the plates.
  10. After 15 minutes, add 50 nanograms of plasmid DNA to the tubes with calcium chloride and bacteria. The amount to be transferred in microliters can be calculated from the known concentration of plasmid DNA. Mix without violence and put back on ice for 10 more minutes.
  11. After 10 minutes, transfer the tubes to the water bath at +42 Centigrades. Do not immerse the tubes in the water. The tip of the tube with the bacteria is kept under the surface of water. The cap should under no circumstance get wet since the water is full of bacteria. Keep exact time and remove the bacteria from the water after 45 seconds.
  12. Keep the tubes on ice for 2-3 more minutes.
  13. Transfer 100 microliters of the contents of the tube to the plates you have marked with a Gilson pipette.
  14. Add 3-5 sterile glass beads and rotate the plate on the surface of the bench to spread the bacteria evenly on the plate.
  15. Seal your plates with a strip of Para film or tape.
  16. Cultivate the plates up side down over night at +37 Centigrades.

Practical day 2: Result of transformation

The results of transformations will be analyzed and biomedical self-tests are discussed. Does anything grow on the plates? The “pimples” on the plates are colonies with millions and millions of bacteria, each and everyone originating from a single bacterial cell. The resulting “transformants”, if any, are re-streaked on fresh plates to ensure that they really are ampicillin resistant. In addition, they can be streaked on plates with a different antibiotic (kanamycin) to see what happens.

Materials:

  • overnight transformation plates, hopefully with colonies of transformants
  • one plate with kanamycin and one plate with ampicillin
  • a disposable inoculation loop

Procedure: restreaking the transformants

  1. Take one ampicillin and one kanamycin plate and divide them with a magic marker (on the bottom) into three equal sectors.
  2. Using a sterile inoculation loop, pick ONE nice colony from the transformation plates and streak it on the plates.
  3. This will result in two identical restreaked plates with the only difference that the other plate contains ampicillin and the other kanamycin.
  4. Cultivate the plate overnight at +37 Centigrad. Keep the plate upside down.
  5. Store the original transformation plates in the refrigerator.

Practical day 3: Results and analysis of results, microscopy

The bacteria are analyzed using UV light with a UV transilluminator (resembling a tanning booth) and with a fluorescence microscope. The groups are allocated time slots for the fluorescence microscopy analysis. Please read the short introduction to microscopy before the practical.

Materials:

  • Glass slides
  • Cover slips
  • Sterile water
  • Sterile inoculation loops
  • Gilsons and tips

Procedure for UV-transilluminator:

  • Wearing protective gear (your skin and your eyes), place your Petri dishes on the UV transilluminator, without the lid.
  • Can you see anything?
  • Which color are the bacteria?
  • Compare these to the plates with the original, untransformed E. coli and your primary transformants.

Microscopy:

  • Prepare a “wet mount” of your bacteria
  1. Get a clean slide and a cover slip.
  2. Smear transformed bacteria in the center of the slide using a sterile loop.
  3. Use a Gilson to add 1 SMALL drop of water to cover the specimen.
  4. Place the edge of the cover slip to the side of the water drop and specimen.
  5. Carefully lower the cover slip over the specimen letting any air bubbles escape.
  6. If you end up with air bubbles, tap the top of the cover slip gently
  7. Dry off any extra water with a piece of paper towel. Remember to throw away the paper in the proper place.
  8. DO NOT GET ANY WATER ON THE MICROSCOPE OR OBJECTIVE!
  9. If the lens is dirty or gets wet, ask for help.
  • The bacteria is studied with fluorescence microscopes.
  • Clean up! Having completed the practical, help the instructors to clean up in the lab.

Discussion instructions and questions for the practical

  1. Go through the whole practical 1, step by step and think about all the different things you did.
  2. Make sure that you understand what happens in every step.
  3. In this practical, you have modified E. coli genetically by transforming it with DNA (a plasmid vector). Do you know of any other ways of modifying organisms genetically?
  4. Why were antibiotics used in the transformation procedure?
  5. What does ampicillin do?
  6. Why is it important to always finish the penicillin treatment?
  7. What does kanamycin do?
  8. Why do we grow bacteria at +37?
  9. Why are bacteria grown on Petri dishes with agar?
  10. What is meant by “rich medium”?
  11. Is E. coli a pathogen? Where can it be found in nature?
  12. The fluorescent protein originally comes from a jellyfish. How can it be active in bacteria?
  13. Will bacterial genes be active in jellyfish? Why yes or why not?
  14. How could you make the gene usable in the human body ( e.g. green fluorescent skin cells for nice fluorescent tattoos)?
  15. How do you think new genes for such fluorescent proteins could be identified and cloned?
Topic revision: r4 - 2009-02-05 - MattiNikkola