Transformation with pGLO

This page is primarily about the techniques of transformation and plating for the pGLO lab. Before you start with this page, you should read these pages for background:

In this lab, you’ll transform E. coli cells with the plasmid pGLO, which contains a gene for green fluorescent protein (GFP). This will allow you to grow bacterial colonies that are bright, fluorescent green. Once you've done that, you can use chromatography and electrophoresis to analyze both the plasmid DNA and the proteins produced by the transformed cells.

Background: Transformation

Bacterial transformation is the process of forcing bacteria to take up DNA from outside the cell. In addition to being an important part of bacterial evolution, transformation is an essential part of gene cloning. Once a recombinant plasmid is created, the plasmid must be inserted into a cell so the plasmid can be reproduced and its genes expressed.

In this lab, you’ll use a simplified transformation protocol using two key treatments. First, you’ll treat the E. coli cells with calcium chloride to cause the membrane to become permeable to DNA. DNA molecules are charged and usually very large; without a treatment to permeabilize the membranes, DNA is unlikely to be taken up. Second, you’ll use a heat shock -- a short incubation at a dangerously high temperature for the cells (42° C). The heat shock will induce a heat shock response in the cells, which means that they will begin producing a number of specialized heat shock proteins, including chaperones and other repair enzymes that have the effect of encouraging the survival of the transformed cells.

Method overview

This lab has multiple parts; you’ll complete it over seven lab periods:

  • Day 1: Transform E. coli cells with pGLO plasmid and plate out the cells.
  • Day 2: Look at the plates to see if your transformation worked (that is, are your bacteria fluorescent?). Pick some transformed colonies and transfer them to liquid cultures. We won't be able to do much more, because we need to grow liquid cultures for the next phase of the lab.
  • Day 3: Use hydrophobic interaction chromatography (HIC) to recover proteins, including GFP.
  • Day 4: Analyze proteins with SDS-PAGE.
  • Day 5: Purify the pGLO plasmid from the transfected bacterial cells.
  • Days 6 & 7: Perform a restriction digest of the plasmid DNA and analyze the restriction fragments by electrophoresis.

This page only covers day 1 and 2. Some parts of the pGLO lab will overlap with the conjugation lab.

Day 1: Transformation

You’ll take a colony of bacteria, add some calcium chloride and pGLO plasmid, then heat shock the cells in an effort to make them take up the plasmid. This process is called transformation. Meanwhile, as a control, you’ll do the same thing with cells but no plasmid. Thus, you’ll have two cultures: + pGLO and -pGLO.

After transformation, you’ll spread these two transformation cultures onto LB plates containing various ingredients:

Ampicillin:
Creates a selective medium, allowing only cells containing the plasmid to grow.
Arabinose:
Induces expression of GFP by binding to the protein AraC. Arabinose creates a differential medium, which means that both pGLO and non-pGLO cells can grow, but they look different (only the pGLO cells become fluorescent).
Glucose:
Inhibits GFP expression. For an explanation of this effect, see Campbell, fig. 18.5: Positive control of the lac operon. The GFP operon in pGLO also contains a CRP binding site as shown for the lac operon. The standard LB growth medium does not contain glucose or other added sugar, so the cells would be in the "glucose scarce" situation shown in that diagram.

You should do one +pGLO transformation and one no-pGLO negative control transformation (not really a transformation, since there is no plasmid DNA). In addition do another transformation with mutant pGLO. I will have to explain what this is later, but for now let's say that it results from bizarre and terrifying genetic experiments conducted in the back room by the Special Projects Team.

After transformation, you'll spread your cells on plates as shown below:

  Ingredients in plates    pGLO  Expected results
Plate # amp arabinose glucose    
1 + + + Growth; fluorescence
2 + + Growth; no fluorescence without arabinose.
3 + + + + Growth; no fluorescence. Inhibited by glucose.
4 + No growth; no plasmid, no amp resistance.
5 + Growth without amp, but not fluorescent.
6 + + +mutant We'll see. For this plate, use 500 µl of the transformation mix.

Using this set of experimental treatments should allow you to find out if the cells are alive, if they got transformed, and if arabinose and glucose can control the expression of GFP in the transformed cells. Each of the treatments is designed to answer a specific question. On an exam, you may see some hypothetical plate results and be asked to interpret them. For example, how will you know if the transformation worked? Would you be able to tell even if you didn’t see fluorescent colonies?

Materials for transformation

  • 1 tube containing 10µl of pGLO plasmid (80 ng DNA/µl)
  • 1 tube containing mutant pGLO (volume is unknow, but use all of it)
  • Sterile LB plates, according to the table.
  • 1 liquid culture of E. coli HB101 (this will be your source of bacteria to transform)
  • 1 tube of CaCl2 transformation solution
  • 1 tube sterile LB broth
  • 3 1.5-ml microfuge tubes (for transformation: one for pGLO, one for no pGLO).
  • Metal inoculation loop
  • Plate spinner
  • Pipetmen, tips, beaker for waste tips
  • Tub with ice

Make sure there is a heat block set at 42° C before you start.

Transformation protocol

Labeling tubes & plates

You'll need to label a lot of things this quarter. You should never be using tubes or plates without any label, because you're likely to mix them up. Label as follows:

  • Plates: We don't reuse them; write directly on the plastic petri dish. Write on the bottom, because you might mix up the lids.
  • Plastic microcentrifuge tubes: We don't reuse them; write directly on the plastic tube. Don't put tape on them, because it's likely to come off.
  • Glass culture tubes: We reuse them. Don't write directly on the glass tube or the plastic cap. Label them with tape (on the tube, not the lid), and peel off the tube when you put the used tubes in the used glassware container.

Read the whole method before you start. There are critical time and temperature steps, and you must have everything ready before you start.

  1. Label your three 1.5-ml microfuge tubes + (for  pGLO) +m (for mutant pGLO) and — (for no pGLO). These are your transformation tubes. Don't use the 0.5 ml tubes.
  2. Pipet 250 µl CaCl2 transformation solution into each tube. (Make sure that this solution is completele thawed and mixed first.)
  3.  Add E. coli HB101 cells to the tube. If using starter plates: Use a metal inoculating loop to transfer several small colonies of bacteria from your starter plate into the (+) tube. Spin the loop in the tube until the bacteria are completely dispersed (no chunks). Repeat this procedure to transfer colonies into your other transformation tubes. All three tubes get bacteria, but two get different PGLO versions and one gets no pGLO DNA.) (If using liquid cultures instead of plates: First, swirl the culture tube so the cells are suspended. Pipet 50 µl of liquid culture into each of your transformation tubes.)
  4. Add 10 µl pGLO plasmid solution (probably the entire volume of the tube) to the + tube. Add the entire volume of the mutant pGLO to the +m tube. Don't add anything to the — tube. You should now have three tubes, all with CaCl2 and some bacterial cells, but only two with pGLO plasmid DNA.
  5. Incubate all three tubes on ice for 10 minutes.
  6. While your tubes are on ice, label your plates according to the table above. Also make sure that you have a heat block ready at 42° C (+/-- 2° C) for the next step.
  7. Place your transformation tubes in the 42°heat block for 50 seconds.
  8. When your 50 seconds are up, transfer your transformation tubes immediately back to the ice. Incubate on ice for 2 minutes.
  9. Add 250 µl LB broth to each transformation tube.
  10. Incubate all three tubes at room temperature for 30 minutes.
  11. Stir the transformation mix gently with a pipet tip, then pipet 100 µl of the transformation mix onto each of the plates as shown in the table above. (For plate 6, the mutant pGLO transformation, use 500 µl of the transformation mix.)
  12. Use a metal inoculation loop to spread the transformation mixes on the plates. Be sure to spread the liquid all over the plate so you’ll get isolated colonies. Put your plates in the incubator until next time.

Before you leave

You should have 6 transformation plates in the incubator.

Dispose of your transformation tubes with bacteria in the biohazard trash. The only colonies you need now are those on your plates.

Gloves, paper towels, etc. go in the regular trash (not the biohazard, unless they’re contaminated).

Beakers used for waste tips: dump the tips into the biohazard trash and put the beaker into the dirty-glassware tub on the cart (if there’s not a tub, check with the instructor).

Bad plates that you may have messed up should be put into the biohazard waste can.

Day 2: Interpreting your results

Look at the table you used for setting up your plates. Each plate is an experimental treatment; some of these are controls. The set of experimental treatments is designed to answer specific questions, and for each question you need to compare a result to a negative control.

Did your cells get transformed?
If your transformed (+pGLO) cells grow on ampicillin (plate 1 or 2) and your un-transformed (-pGLO) cells don't grow on amp (plate 4), then you know your transformation worked. The negative control (no amp resistance without pGLO) is essential. Un-transformed cells might grow on amp if they were already amp resistant before you started, or if the "amp" plates didn't actually contain the ampicillin they were supposed to have.
Satellite colonies: Look closely at your plate #1 (pGLO, amp, arabinose). You should see big colonies that are fluorescent. You might also see smaller colonies that aren't fluorescent. Those are satellite colonies, and they don't contain the plasmid.
IMG 2684 reduce
The large, fluorescent, pGLO-transformed colonies are producing and secreting  β-lactamase, an enzyme that breaks down ampicillin. The transformed colonies eventually create an ampicillin-free zone surrounding each colony. The satellite colonies don't have the plasmid or the amp resistance, but they grow in the amp-free zone. Satellite colonies are usually avoided by incubating plates no more than 24 hours, but for the 6B lab we need to incubate 48 hours to accommodate our schedule. When you are taking colonies from your plates for the next steps, be sure to avoid the satellite colonies.
Does arabinose control GFP expression?
If the pGLO-transformed cells are fluorescent on arabinose (plate 1) and non-fluorescent without arabinose (plate 2), arabinose induces fluorescence.
Does glucose inhibit GFP expression?
If pGLO-transformed cells are fluorescent on arabinose (plate 1) but not on arabinose + glucose (plate 3), glucose inhibits GFP expression?
Is pGLO necessary for the fluorescence?
If transformed cells are fluorescent on amp (plate 1) but non-transformed cells are not fluorescent (plate 5), pGLO is necessary for fluorescence.
What about that mutant pGLO?
We created the mutant versions of pGLO in our lab using a process called site-directed mutagenesis to alter the GFP coding region. The mutant versions should still function as a plasmid, producing amp-resistant colonies. The expected mutations are in the GFP gene, perhaps altering the color of the colonies. Do you see colonies that either have fluorescence of a different color or lack fluorescence altogether? If the colonies aren't fluorescent, do they contain a plasmid?

Next steps

After viewing and recording your results, you will use your transformed cells both for DNA analysis (Total Nucleic Acid Extraction, Plasmid Purification and Restriction Digest) and for protein analysis (Hydrophobic Interaction Chromatography and SDS-PAGE). Thus, you need to save some transformed colonies.

Restreak some plates

Depending on your plate results, you might want to restreak some bacteria from one of your transformation plates onto a new plate. (Check with your instructor to see whether you should do this today.) This will give you additional fresh colonies to use in later labs. Use the technique from starting bacterial cultures. For this lab, you should restreak on a plate with ampicillin to ensure that only cells containing the pGLO plasmid will grow. Arabinose is optional in this plate, but might be helpful; discuss this with the instructor. Once you restreak a plate, put it in the incubator so it can grow. Do not seal this new plate with tape.

Save some of your transformation plates

In addition to the newly restreaked plate described above, you should save some of your transformation plates to make sure you have colonies for later use. If your results are good, save plate 1, which has fluorescent pGLO-transformed colonies. (If you don't have good colonies on that plate, you may be able to use another plate that has colonies containing pGLO; this could be any plate with ampicillin.)

Also save plate 6. This plate may contain colonies with different types of pGLO, producing green, blue, or non-fluorescent colonies.

Review

Terms and concepts

You should be able to explain the roles of all these things in this experiment.

  • Ampicillin
  • Arabinose
  • AraC
  • Coding region (or coding sequence)
  • Constitutively expressed
  • Glucose
  • Operon
  • Origin of replication
  • Origin of transfer
  • Plasmid
  • Promoter
  • Restreaking
  • Satellite colonies
  • Selective medium and differential medium

Review questions

  1. Why is it important to use ampicillin in this experiment?
  2. Compare & contrast the GFP operon with the lac and trp operons (see Campbell for a discussion of lac and trp; these will be covered in lecture.)
  3. What is the purpose of plates 4 & 5, which have no pGLO?
  4. How do you know if the transformation worked? (Hints: you need to look at an experimental plate vs. a control plate, and it's not necessarily about fluorescence?
  5. How do you know if arabinose does what it's supposed to do? (Again, think in terms of experimental plate vs. control plate.)
  6. How do you know if glucose does what it's supposed to do? (Again, think in terms of experimental plate vs. control plate.) Why does it make sense that glucose  would affect GFP expression?
  7. Suppose you wanted to make a version of pGLO that can be transferred by conjugation. How would you do it?
  8. Suppose you wanted to add the GFP gene to the pARO180 plasmid. How would you do that?
  9. What are satellite colonies? How might they affect your results in this experiment? How could you have avoided satellite colony formation?

 References & further reading

Background

pGLO Map and Resources from Bio-Rad.

Transformation protocols

Transformation of Escherichia coli Made Competent by Calcium Chloride Protocol. Tu, 2008. American Society for Microbiology. Excellent description of the classic protocol.

Transformation videos

How to perform a bacterial transformation from Bio-Rad Explorer. 7 minutes. Good, clear video that is specifically about the pGLO experiment. We do some slight variations on this in 6B, so don't follow the video instructions exactly.

Competent Cell Transformation from Invitrogen.

How to Transform E. coli by Heat Shock from Synthetic Biology One.

Bacterial Transformations from Addgene.

A- A A+