Laboratory Experiment #7 - Mr. Green Genes

The first component of the Lycoming College Molecular Biology and Bioinformatics Project Jeffrey D. Newman, Lycoming College © 1999

Introduction – week 1

In this experiment, you will explore several fundamental techniques that have revolutionized biology! While incomplete, the recently acquired understanding of how organisms function at the subcellular level has changed the way scientists approach biological questions. Molecular Cell Biology has touched every corner of biology. Specific examples include the use of pre-implantation diagnosis and gene therapy in the medical field; the design of new, more specific drugs with fewer side effects; engineering of food crops to confer disease, frost, and drought resistance; genetic modification of bacteria for bio-remediation of polluted sites; and genetic analysis to determine evolutionary relationships between organisms or to track the migration of specific populations over time.

We will begin the exercise by learning how to isolate and manipulate the genetic material, DNA. The specific DNA we are interested in comes from the bioluminescent jellyfish Aequorea victoria. This DNA contains the jellyfish gene that encodes the instructions for a green fluorescent protein (GFP) that causes the jellyfish to fluoresce and glow in the dark.

The easiest way to work with DNA is to let bacteria do all the work for you. It is impractical to chemically synthesize large amounts of a specific DNA molecule, although it can be done. A better strategy is to place the specific DNA molecule into bacteria and provide nutrients that allow the bacteria to reproduce themselves and the foreign DNA to a density of >10⁹/mL. One small complication, however, is that the jellyfish GFP gene does not have the right signals to be copied by bacteria. This problem can be overcome by inserting the GFP gene into another piece of DNA, a plasmid, that can be copied because it contains a bacterial origin of replication (ori).

In addition to one large circular chromosome, bacteria often contain one or more small, circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that are beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The recent occurrence and spread of bacterial resistance to multiple antibiotics is due to the transmission of plasmids.

Discuss the following questions within your group (4).

What are the major components (macromolecules) of cells?

How is DNA chemically different from the other components of cells?

How could these differences be used to purify plasmid DNA from other cellular constituents?

Compared to many other areas of biology, the technical skills required to do molecular biology are very simple; the key is to get comfortable working with small volumes. These small volumes are measured out using micropipettors. A "set" consists of three pipettors that together can accurately measure and dispense volumes from 0.5 mL to 1000 mL (1 mL).

• Volumes between 0.5 mL and 20 mL are measured using the smallest pipettor, the I-20. The yellow digit on the bottom of the setting display indicates tenths of a microliter, e.g. setting of " 0 - 5 - 3 " corresponds to 5.3 mL.
• Some sets have I-100’s and some have I-200’s for the intermediate volumes between 20 mL and 100 or 200 mL. The 3 digits on the display show hundreds, tens and ones; e.g. " 0 – 7 – 5 " corresponds to 75 mL.
• The largest pipettor, a I-1000, is used for volumes between 100 and 1000 mL. The yellow digit at the top of the setting display indicates thousands of mL;
  e.g. " 0 – 8 – 0 " corresponds to 800 mL (or 0.80 mL).

Your lab instructor will demonstrate the use of the pipettors.

1. Set dial to desired volume. Do not try to turn volume setting dial beyond the maximum volume for the pipettor. This is how students most frequently damage the pipettors.
2. Attach a pipette tip. The small yellow or white tips are used on the I-20, I-100 and I-200’s; the large blue tips are used with the I-1000 pipettors. Be sure tip is attached securely to pipettor, liquid will drip out if seal is not tight.
3. Press plunger down to 1^st stop. Place tip into liquid
4. Slowly release plunger so that liquid is drawn into pipette tip. Always hold pipettors with the shaft pointing down, especially when liquid is in the tip. If liquid does get into the shaft, please tell your instructor immediately, so that it can be cleaned out. Damage and corrosion can occur if salt solutions dry inside pipettor.
5. Dispense liquid into desired tube by pushing down plunger to the second stop. Remove tip from liquid before releasing plunger, otherwise you will draw liquid back into tip.
6. Eject used tips into coffee cans at your bench.

Practice using the pipettors to measure the following volumes of water; 2 mL, 12 mL, 32 mL, 100 mL, 350 mL, 900 mL. How could you tell whether your measurements were accurate?

Each pair of students has been provided with a culture of E.coli carrying a plasmid with the GFP gene. Use the following procedure to purify the plasmid DNA from the E.coli cells. The location of the plasmid DNA at each step is shown in bold, so you don’t throw away the tube with your DNA by mistake

Plasmid DNA minipreps - CTAB method

Reference: Del Sal G, Manfioletti G, Schneider C (1989) BioTechniques 7:514-520 The CTAB-DNA precipitation method: A common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing.

Procedure (students should work in pairs):

1. Label two 1.5 mL microfuge tubes with your initials. Using a disposable plastic pipette, fill the two tubes with an O/N culture of E. coli containing the pGLO plasmid. Close the tubes.
2. Place the cultures in the centrifuge on opposite sides of the rotor for balance. Always place tubes in the centrifuge with the hinge pointing up; the resulting pellet (which is sometimes difficult to see) will always be located on the back wall of the tube. Centrifuge tubes for 30 sec. Pour supernatant into waste cans at your bench, tap tubes containing cell pellets on paper towel to remove any remaining liquid.
3. Add 175 mL STET buffer to each tube, vortex to thoroughly resuspend cell pellets (no chunks).
4. Add 12.5 mL lysozyme solution (10 mg/mL) to resuspended cells, vortex briefly, place in a boiling water bath for exactly 1 min. Immediately centrifuge for 10 min. (Don’t forget to balance tubes and put the hinges up).

While the samples are spinning, discuss the following questions within your group.

STET Buffer contains:

• 8% Sucrose - osmoticum
• 20 mM Tris-HCl pH 8.0 - buffer
• 50 mM EDTA (ethylenediaminetetraacetic acid) – binds (chelates) metal ions
• 0.1% Triton X-100 – detergent

Lysozyme is an enzyme that breaks down bacterial cell walls.

What do you think is accomplished in these first few steps?

How does each component contribute to this goal?

Lysozyme is abundant in tears, what is its probable function for the body?

5. Use a toothpick to remove pelleted cell debris from the tube. Discard the pellets, but save the supernatants. What do you think is present in the supernatant?
6. Add 10 mL RNaseA (5 mg/mL), vortex briefly, incubate for 10 min. in 68^oC heating block.
7. Add 10 mL CTAB solution, vortex briefly, leave at room temp for 3 min.
8. Centrifuge at room temp for 5 min., carefully remove supernatant with a pipette and discard supernatant, save the pellets.
9. Add 150 mL 1.2 M NaCl to each tube; dissolve the pellets by vortexing. Combine the two samples into a single tube by transferring the contents of one tube into the other.
10. Add 750 mL ice-cold 95% EtOH, vortex, place on ice for 15 min.

RNaseA is an enzyme that degrades RNA; cetyltrimethylammonium bromide (CTAB) forms an insoluble complex with DNA at low [NaCl], but releases DNA and is soluble at high salt concentrations.

Brainstorm within your group about how steps 6-10 contribute to the purification of the plasmid DNA.

Assemble a flow chart showing the main accomplishments during the purification procedure.

11. Centrifuge sample tube for 10 min. to pellet DNA. Pour supernatant into waste cans at your bench, tap tube containing DNA pellet on paper towel to remove any remaining liquid.
12. Rinse DNA pellet by adding 500mL 70% EtOH, close cap, gently swirl ethanol around inside of tube to wash walls, decant well over a paper towel. Be careful not to lose DNA pellet.
13. Dry DNA in centrifugal evaporator (speed vac) for about 5 minutes.
14. Add 50 mL TE buffer to dried DNA pellet, vortex well. Store at -20^oC.

Introduction - Week 2

Last week, you performed a series of steps designed to isolate DNA from E.coli. How do you know whether you have DNA in the tube? How do you know whether it’s the right DNA? These questions can best be answered by physical analysis. How do DNA molecules differ? How is the DNA in your cells different from that of your lab partner?

To determine whether we have the right DNA, we must know what to look for. In the beginning of this lab, it was pointed out that the jellyfish GFP gene was present within a circular bacterial plasmid. How did it get there? Genetic Engineering! This powerful technology allows scientists to cut separate pieces of DNA, such as the GFP gene and the plasmid, and paste them together to make a recombinant DNA molecule. By re-cutting the DNA at the sites where it was originally pasted, we can examine the fragments to see if they are same DNAs that were put together.

Proteins called restriction endonucleases recognize specific short DNA sequences and cut the DNA at those sequences. For example, the restriction enzyme EcoRI from the bacterium E.coli recognizes and cuts the sequence GAATTC. When a restriction enzyme is incubated with a piece of DNA containing the specific recognition sequence, the DNA is cut into fragments. To identify specific fragments, one must separate the fragments based on their size. This is accomplished by gel electrophoresis. Since DNA is negatively charged (due to the phosphate groups in its backbone) it is attracted to the positive electrode when subjected to an electric field. If the DNA is present within a gel, its movement toward the positive electrode will be hindered to different extents, depending on the size of the DNA molecule (would a fish swim better in water or jello?). Thus, one can use the distance migrated by a particular DNA fragment to determine the size of the fragment.

Restriction enzyme digestion and gel electrophoresis can provide information about physical characteristics of a particular DNA such as the size and relative position of specific base sequences. However, these do not provide information on the function of the DNA. To examine the function of a specific DNA, it must be studied in vivo (in life). What is the function of DNA?

DNA contains genes encoding proteins (usually) that do something for the organism. Two genes present on the pGLO plasmid encode a beta-lactamase enzyme and the green fluorescent protein. Beta-lactamase is an enzyme that breaks down members of beta-lactam family of antibiotics, which includes ampicillin, penicillin and amoxicillin. These antibiotics are used to treat infections because they normally kill E.coli and other bacteria. However,

when a plasmid such as pGLO is introduced to bacteria, either intentionally or unintentionally, the bacteria express the beta-lactamase gene, breakdown the antibiotic, and therefore are not killed – they are now resistant to the antibiotic!

The process by which free DNA is taken up by and is incorporated into an organism is called genetic transformation. An organism with such DNA is referred to as transgenic. Transgenic plants, animals, yeast and bacteria have been become critical tools for the pharmaceutical, agricultural, and biomedical industries.

Transforming plasmid DNA into bacteria is relatively simple. While the bacterial cell wall is porous, so that DNA can pass through it easily, the plasma membrane is more of a challenge. Preparation of cells for transformation involves incubation with calcium (Ca²⁺ ) to neutralize the negative charges that would cause repulsion of the negatively-charged DNA. Bacteria are also incubated on ice to induce the low temperature adaptation of increasing membrane permeability. Such cells are now "competent" to take up DNA. At this stage, a brief heat shock opens holes in the membrane, which allows the plasmid DNA to enter the cell. Fewer than 1% of the bacterial cells will actually take up DNA, so a method is necessary to identify those that have. Spreading the bacteria onto an agar plate containing the antibiotic ampicillin kills those cells that have not acquired the plasmid with the beta lactamase gene. Only the bacteria that have taken up the plasmid will be able to multiply. Since they are on an agar surface, this multiplication will form a visible "pile" or colony of bacteria on the plate.

A final concept demonstrated by this lab exercise is gene regulation. If all of the cells in your body have the same set of genes, why don’t your brain cells produce the same digestive enzymes that your stomach cells produce?

All organisms regulate the expression of genes, in other words, they are able to control when specific genes are turned on and when they are turned off. In higher organisms, such as plants and animals, the primary function of gene regulation is cell and tissue specialization. Different types of cells express different sets of genes (and therefore produce different proteins) at different stages of development. In bacteria, the primary function of gene regulation is adaptation to the environment. The production of proteins is an energetically expensive process. It would be a huge waste of energy for a cell to produce the proteins necessary to breakdown a particular nutrient if the nutrient was not available. The expression of genes is generally controlled by the binding of proteins to a region in front of the gene called a promoter.

When the jellyfish green fluorescent protein gene was inserted into the plasmid, it was placed after a promoter that normally controls the expression of genes involved in metabolizing the sugar arabinose. When arabinose is absent, the promoter is "off" and the GFP gene is not expressed, i.e. the protein is not produced. In the presence of arabinose, the promoter is "on", which allows the GFP gene to be transcribed into RNA. The RNA is translated to produce the protein, which causes the bacteria to glow green.

Procedure – for a group of 4 students, except where noted. During the incubations described in steps 4 and 5, the computer-based analysis of DNA sequence data will be demonstrated for you.

1. Begin preparation of competent E. coli cells as follows:

1. Use a sterile disposable pipette to fill two 1.5 mL microfuge tubes with mid-log phase culture of E. coli. Centrifuge at 7000 rpm for 30 seconds.
2. Decant supernatant into the waste can, save the pellets. Resuspend each cell pellet in 1 mL ice-cold 0.1 M CaCl₂ by vortexing. Incubate cells on ice for 30 min.

2. Set-up restriction enzyme digestions of plasmid DNA: (each pair of students)

1. Add 5 mL of DNA to each of the 3 tubes provided to you. One tube (B) contains the restriction enzyme BamHI, another (X) contains the enzyme XhoI, and the third (T) contains only TE buffer.
2. Label the tubes with your initials and incubate in the 37^oC heating block for 45 min.

3. Prepare a 0.8% agarose gel as follows:

1. Securely tape ends of gel tray such that a small amount of tape is on underside of tray. Place tray on a sheet of plastic wrap in case of leakage. Align comb in tray parallel with and 1 - 1.5 cm from the end of the tray.
2. Add 0.32 g agarose to 40 mL water in a 125 mL erlenmeyer flask, heat mixture in microwave on high setting for 1 - 1.5 min, or until mixture begins to boil.
3. Using a folded paper towel to hold the neck of the erlenmeyer flask, swirl the gel mixture well, and return to microwave. Heat for an additional 30 - 45 sec, or until mixture begins to boil again.
4. Carefully remove molten gel solution from microwave using a folded paper towel to hold the neck of the erlenmeyer flask.
5. Add 0.8 mL 50x TAE buffer, 20mL 1 mg/mL ethidium bromide (final conc = 0.5 mg/mL), swirl to mix, pour into gel tray, allow to stand at room temp for 20 - 30 min to solidify. Note: Ethidium bromide is a carcinogen! Gloves must be worn when working with gels and gel buffers.

4. Complete preparation of competent cells, begin transformation.

1. Centrifuge cells suspended in CaCl₂ at 7000 rpm for 30 seconds, decant supernatant save the pellets. Resuspend each cell pellet in 100 mL ice-cold 0.1 M CaCl₂ by vortexing.
2. Add 1mL of each DNA preparation to the same tube of resuspended cells, incubate both tubes on ice for 30 min.

5. Heat shock cells for transformation.

1. Place transformation tube in 42^oC heating block for 2 minutes.
2. Add 1 mL LB liquid, mix by gently inverting tube several times.
3. Lay tube on its side in 37^oC incubator for 30 min.

6. Plate out transformation

1. After incubating for 30 min at 37^oC, invert transformation tubes several times to suspend cells.
2. Pipette 100 mL of transformation mix onto each of the following plates: LB + arabinose, LB + ampicillin, LB + ampicillin + arabinose. Light bunsen burner, remove glass spreader from beaker of ethanol, pass through flame to ignite ethanol. After ethanol has burned off spreader, spread cells over plates. Incubate plates in the inverted position overnight at 37^oC.

Day 3

1. Load samples into agarose gel, begin electrophoresis.

1. Fill gel chamber with 1x TAE buffer such that the level of liquid just covers center platform. Carefully remove the comb and tape from ends of gel, place gel in chamber with the wells near the negative electrode (anode-black), add sufficient 1x TAE to just cover the gel. Save the plastic wrap for later.
2. Cut a small piece of parafilm and place on bench near gel, "spot" a 1-2 mL aliquot of loading dye onto parafilm for each sample to be loaded on gel.
3. Draw sample (10 mL) into a pipette tip, pipette up and down onto a spot of loading dye to mix, load sample into well of gel. Be careful not to poke pipette tip through bottom of well.
4. Between the samples from the two pairs of students on each gel, load 10 mL of the DNA size marker – Lambda phage (virus) DNA cut with the restriction enzyme BstEII.
5. Place cover over gel chamber, turn on power supply and set to 120 V, press run button. Confirm proper operation by checking for gas production (bubbles) at electrodes (electrolysis of water).

2. Observe transformation plates in normal light, and when exposed to long-wave ultraviolet light. Record your observations. Count the numbers of colonies on each plate (if possible), estimate the percentage that glow green on each plate. Discard plates in the autoclave bag on the side bench.

3. Examine electrophoresis gel.

1. When the fastest migrating dye has migrated over half the length of the gel, turn off power supply.
2. Wear gloves to remove gel tray from the chamber. Slide gel off tray onto the plastic wrap.
3. Place gel on UV transilluminator, view and photograph gel. **Caution – do not look directly at transilluminator surface, except through UV-blocking cover.

Data analysis

Gel Electrophoresis

1. Measure the distance (in mm) migrated by each DNA fragment on your gel by measuring from the front (leading edge) of the well to the front of each band and enter the data for the Lambda BstEII marker DNA in the table below. The migration of linear DNA through a gel shows an inverse, exponential relationship with its size (mass, length). On the graph paper provided, plot three graphs, each with migration on the X-axis:

1. Length vs. migration on standard graph paper
2. Length vs. migration on semi-log paper
3. Log of length vs. migration on standard graph paper

Draw a straight line through the data points on graphs b and c (points at the extremes probably will not fall on the line due to a limited range of effective separation by agarose and measurement errors). These are your standard curves.

Migration of DNA bands after BstEII-digestion of 1000 ng of lambda DNA.

Use the standard curve to estimate the sizes of the pGLO DNA fragments.

2. The green fluorescent protein is 229 amino acids long. How large a piece of DNA would be required to encode this protein? Are any of the fragments you obtained near that length?
   
3. It is easy to understand why multiple bands would be present in the lanes containing DNA that had been cut with a restriction enzyme, but why do you think there are several bands present in the lane containing undigested plasmid DNA?

Transformation

1. Which plates should be compared to determine whether genetic transformation has occurred? Why?
   
2. What is meant by control plate? What purpose does a control serve?
   
3. How much bacterial growth do you see on each plate? If you observe any difference, what might be the cause of this?
   
4. What color are the bacteria under white light? Under long wave UV light? Explain any differences between the plates.

Materials for a lab section of 24 students:

Day 1

• 1 70^oC heating block
• 1 freezer box
• 1 boiling water bath (hotplate, 1 L beaker, "bubble rack")
• 3 high speed microcentrifuges (1 per bench)
• 3 vortexors
• 6 jars sterile 1.5 mL microfuge tubes
• 6 sets (P-20, P-100/200, P-1000) of micropipettors (1 set at each end of bench)
• 6 boxes blue tips, 6 boxes yellow tips
• 6 small coffee cans
• (6) 250 mL beakers containing toothpicks, disposable plastic pipettes
• (6) 1.5 mL tube with 0.1 mL cetyltrimethylammonium bromide (CTAB) solution 10% wv in dH₂O. *CTAB will usually precipitate after each use, warm to 37^o to redissolve.
• 6 test tube racks, each with:
• (2) 3.0 mL cultures of E.coli/pGLO in LB Amp (100 m g/mL)
• 10 mL 1.2 M NaCl in 15 mL plastic tube
• 10 mL dH₂O
• 10 mL STET in 15 mL plastic tube (0.1 mL Triton X-100; 10.0 mL 0.5M EDTA pH 8.0; 5.0 mL 1.0M Tris-HCl pH 8.0; 8.0 g Sucrose; 84.9 mL dH₂O)
• 6 styrofoam containers of ice with
  • 10 mL 95% EtOH in 15 mL plastic tube
  • 10 mL 70%EtOH in 15 mL plastic tube
  • 100 m L RNaseA in TE (5mg/mL) in 1.5 mL microfuge tube (heat for 10 min. in boiling water bath after preparation to destroy DNase).
  • 100 m L Lysozyme in TE 10 mg/mL in 1.5 mL microfuge tube
  • 100 m L TE buffer
• 12 microfuge tube racks
• 12 fine tip marker pens

Day 2

• 1 37^oC incubator , 1 37^oC heating block, 1 42^oC heating block
• 1 gel prep station
  • digital scale (0.01 g precision), weigh boats
  • styrofoam rack with agarose, 50X TAE, ethidium bromide (1.6 mg/mL)
  • dH₂O carboy
  • 50 mL or 100 mL graduated cylinder
  • 1 box each of small, medium, large gloves
  • coffee can for waste
  • microwave
• 3 microcentrifuges (1 per bench) 3 vortexors
• 3 boxes plastic wrap
• (6) 250 mL beakers containing toothpicks, disposable plastic pipettes
• 6 spreaders in 200 mL beakers with 1 cm 95% ethanol
• 6 boxes blue tips, 12 boxes yellow tips, 6 small coffee cans
• 6 jars sterile 1.5 mL microfuge tubes 6 bunsen burners and lighters
• 6 test tube racks, each with:
• 3.0 mL log phase culture of E.coli in LB liquid
• 3.0 mL LB liquid
• 6 styrofoam containers of ice with
  • 10 mL 0.1 M CaCl₂ in 15 mL plastic tube
  • (2) 0.5 mL microfuge tubes labelled "T", containing 5 m L TE buffer
  • (2) 0.5 mL microfuge tubes labelled "X", containing 5 m L XhoI mix
  • (2) 0.5 mL microfuge tubes labelled "B", containing 5 m L BamHI mix
  • (Mixes prepared for class - 35 m L H₂O, 10 m L buffer, 5 m L enzyme)
• 6 gel setups (gel tray, comb, tape, 125 mL erlenmeyer flask)
• 12 sets of micropipettors (I-20, I-100/200, I-1000)
• 12 microfuge tube racks
• 12 fine tip marker pens
• 12 LB agar plates
• 12 LB + amp (100 m g/mL) agar plates
• 12 LB + arab (5 mg/mL) agar plates
• 12 LB + amp (100 m g/mL) + arab (5 mg/mL) agar plates

Day 3

• 1 transilluminator, 1 camera with 2 boxes film
• 3 hand held UV lights
• 3 1 L flasks with 0.75 L 1X TAE
• 3 boxes plastic wrap
• 12 sets (I-20, I-100/200, I-1000) of micropipettors
• 6 boxes blue tips, 12 boxes yellow tips
• 6 pieces parafilm (2 cm x 5 cm)
• 6 small coffee cans
• 6 gel setups - power supply + gel chamber,
• 6 styrofoam containers of ice with
  • 50 m L l BstEII DNA marker in 0.5 mL microfuge tube
  • 200 m L 6x gel loading dye
• 12 rulers (mm scale)

This page was created or last modified on 8/4/99 by Jeff Newman
and has been accessed  times since 8/1/99.

© 1998, 1999  Jeffrey D. Newman