pGLO

The Bio 6B lab explores bacterial plasmids and operons through a set of connected experiments over multiple lab days. The concepts behind these labs are presented in a set of related pages on this site:

In addition, there are multiple pages for the experimental methods, which you'll find in the menu.

 

Background

In Bio 6B, you'll work with the plasmid pGLO in a long series of experiments, using multiple techniques of molecular biology. This recombinant plasmid, created by researchers at Bio-Rad, combines a gene for green fluorescent protein (GFP), cloned from a jellyfish, with control elements copied from a bacterial operon. The end result is a system that allows for bacterial expression of a eukaryotic gene. You'll be able to transform E. coli cells with the plasmid and manipulate the conditions to control whether the cells express the protein, and you'll be able to see which bacterial colonies are producing it. The pGLO plasmid offers us an experimental system for working with a wide range of concepts and techniques in molecular biology.

Green Fluorescent Protein

As the name implies, GFP is green and fluorescent; if you shine an ultraviolet light on a colony of bacteria that are producing this protein, you’ll see the colony glowing bright green. The GFP absorbs the short-wavelength, high-energy photons from the UV, and re-emits longer-wavelength, lower-energy green photons.

The gene for GFP originally comes from the jellyfish Aequoria victoria. This remarkable protein has turned out to be useful in research, and researchers have tweaked it to make it even more fluorescent. GFP is often used as a reporter gene; by connecting GFP to another gene so both are expressed together, it becomes possible to see where both the GFP and the gene of interest are expressed. There are thousands of research papers based on this approach.

Fluorescent proteins are common in marine invertebrates (especially cnidarians), although the reasons for this are not clear. It's possible that fluorescence is important for communication or camouflage in the deep sea. Green Fluorescent Proteins also provide protection against superoxide radicals by converting the reactive oygen species into less harmful forms. It's not clear whether this is an important factor in the evolution of these proteins.

The GFP operon 

The pGLO plasmid was engineered to allow inducible expression of GFP. “Inducible” means that the cells will only produce GFP when an inducer (in this case, the sugar arabinose) is added. This level of control was achieved by connecting the GFP coding region with a bacterial promoter. If the entire jellyfish GFP gene, including its eukaryotic promoter, had been inserted into the plasmid, the gene wouldn't be expressed in bacteria.

AraBAD operon compared to GFP operon.

The GFP operon in pGLO uses the pBAD promoter from E. coli. In wild bacteria, the pBAD promoter controls the expression of the AraBAD operon, which encodes genes coding for proteins (called AraB, AraA, and AraD) involved in catabolism of the sugar arabinose. Those enzymes would only be expressed when arabinose was present, similar to the lac operon. The AraBAD mRNA has three open reading frames (ORFs), encoding the three proteins in the  arabinose pathway. In pGLO, the coding regions for arabinose-digesting enzymes have been replaced with the GFP coding region, so the GFP operon includes only one ORF.

This represents a common approach in genetic engineering: an existing promoter is used to regulate a new gene. The goal in developing the pGLO plasmid was to create a system that would allow GFP expression to be controlled. When arabinose is added to E. coli cells that contain pGLO, the GFP gene will be highly expressed, becoming one of the most abundant proteins in the cell. When arabinose is not present, the amount of GFP expression will be close to zero.

The AraBAD & pGLO operons have multiple regulatory sites

In addition to the promoter, this operon has multiple sites where regulatory proteins (AraC and CRP) bind and upregulate or downregulate transcription.

Structure of the AraBAD operon.

Note that there are two different transcription units (regions of DNA that get transcribed). One is GFP (in the place of AraB, AraA, and AraD). The other is AraC, which is a regulatory protein that controls the expression of the operon. The arrows on the transcription units represent the direction of transcription. Transcription goes in different directions for these two transcription units, because they are transcribed from different strands of the DNA. The promoter for GFP is called pBAD (or simply the AraBAD promoter), and pC is the promoter for AraC.

Don't try to memorize the names of all these sites; read on to see how they work.

AraC represses transcription when arabinose is absent

Like the lac operon, the AraBAD operon is negatively controlled by a repressor protein. When no arabinose is present, the repressor protein (called AraC in this case) binds to operator regions in the DNA and blocks transcription of the operon.

AraBAD operon repressed by AraC protein blocking RNA polymerase.

In the absence of arabinose, the AraC repressor protein binds in several locations on the DNA, holding the DNA in a loop that prevents RNA polymerase from binding to the promoter. (In the diagram, the line represents the double-stranded DNA.) AraC proteins form a homodimer: two identical proteins bound together. Since each AraC is bound to a different site on the DNA, the DNA is looped. In this situation, AraC is acting as a negative regulator of transcription (in other words, a repressor).

Note that AraC represses transcription of both the GFP gene and the AraC gene itself. (In other words, when the level of AraC protein builds up in the cell, it turns off the AraC gene, so no more AraC protein is synthesized until the amount of AraC protein decreases. Therefore, there is always a small amount of AraC protein present.)

AraC displays several characteristics that are common in proteins that regulate gene expression:

  • Binds to specific DNA sequences. Gene expression is regulated by protein-DNA interactions.
  • Alters conformation of DNA (forming the loop). This is different from the lac and trp operons, but is common in eukaryotic gene regulation.
  • Acts as a dimer (two copies of the protein bound together). Many transcription factors, as well as restriction enzymes, also work this way.
  • Is allosterically regulated, controlling its ability to bind to DNA.

AraC upregulates transcription when arabinose is present

Arabinose acts as an allosteric regulator of AraC, changing which DNA sites it binds to and how it forms a dimer.

AraC regulation

Remember that arabinose is the sugar that gets catabolized by the proteins of the AraBAD operon. When arabinose is added to the environment in which E. coli live, it binds tightly to AraC. The AraC protein lets go of one of its former binding sites and attaches to another. In this diagram, the two copies of AraC (with the green "A" arabinose molecules attached) sit side by side on the DNA; in fact, they are stuck together as a new kind of dimer. In this position, AraC no longer acts as a repressor. In fact, AraC now upregulates transcription by helping to stabilize RNA polymerase on the promoter. In eukaryotic gene regulation, a protein that attaches near a promoter and assists RNA polymerase is called a transcription factor. For some reason, that term is less commonly used for bacterial genes, but I think it fits AraC perfectly in this situation.

To summarize, the effect of AraC completely changes when arabinose is added:

  • Arabinose absent: AraC acts as a repressor, downregulating transcription (negative gene regulation).
  • Arabinose present: AraC acts as a transcription factor, upregulating transcription (positive gene regulation).

CAP upregulates transcription

The Catabolite Activator Protein, CAP (also called cyclic AMP receptor protein, CRP), also helps regulate the AraBAD and GFP operons. You encountered this protein acting on the lac operon. The AraBAD operon, like the lac operon, encodes proteins for catabolizing an uncommon type of sugar. In both cases, glucose is the preferred energy source, because fewer enzymes are required. When E. coli cells are starved for glucose, and their energy stores are low, they produce cAMP. In both the lac  and AraBAD/GFP operons, high cAMP level allosterically activates CAP, which binds to the promoter, helping RNA polymerase start transcription.

For maximum expression of the GFP gene in pGLO, you'll need to culture your bacteria in plates with arabinose and no glucose. You'll be able to see how strongly glucose affects GFP expression by looking at your pGLO plates grown with and without glucose added to the medium.

Other pGLO features

Here is a map of the whole pGLO plasmid:

pGLO restriction map

In addition to the GFP operon, you should understand two other elements of the pGLO plasmid:

  • bla — also known as the ampicillin resistance gene; a gene that encodes the enzyme β-lactamase, which breaks down the antibiotic ampicillin. The bla gene, like all genes, includes a promoter and a coding region. This gene is constitutively expressed, meaning that it's continually produced at a low level.
  • ori – the origin of replication. DNA replication starts here. Every plasmid has an origin of replication; useful plasmids such as pGLO and pARO180 contain high copy number origins, meaning that the origin causes the plasmid to be replicated frequently, so that each cell will contain hundreds of plasmid molecules.

Size: the pGLO plasmid is 5371 bp. The GFP coding region is about 714 bp, and the GFP protein is about 26 kD, with 238 amino acids. This will become relevant when you analyze the protein and DNA on gels.

pGLO mutants

We also have some mutant versions of pGLO in the lab. A group of Bio 6B Special Projects students used PCR mutagenesis to alter the nucleotide sequence of the GFP coding region. One of the mutant versions of the plasmid ends up producing a Blue Fluorescent Protein, and another produces no fluorescent protein at all. You'll be able to investigate the sequences to understand why in a bioinformatics exercise later this quarter.

 Review

Terms & concepts

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

  • Allosteric regulation
  • Ampicillin
  • Arabinose
  • AraC
  • AraBAD operon
  • Coding region (or coding sequence)
  • Constitutively expressed
  • CAP (Catabolite activator Protein); also called CRP (cAMP Receptor Protein)
  • Dimer and homodimer
  • Glucose
  • Inducible operon
  • Operon
  • Origin of replication
  • pBAD promoter
  • Plasmid
  • Promoter
  • Satellite colonies
  • Selective medium and differential medium
  • Transcription unit
  • Upregulation vs. downregulation. Anything that increases gene expression is upregulation, so inactivating a repressor (removing negative regulation) would count as upregulation. These terms are used because gene expression isn't normally just on or off; genes are regulated by degrees.

Don't worry about memorizing the names of all the binding sites in the DNA such as I1 or O1; just focus on what they do.

Review questions

  1. Compare and contrast the GFP and AraBAD operons. How many ORFs does each have?
  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. Suppose you wanted to make a version of pGLO that can be transferred by conjugation. How would you do it?
  4. Suppose you wanted to add the GFP gene to the pARO180 plasmid. How would you do that?
  5. Why is it important to use ampicillin in this experiment?
  6. What are satellite colonies? How might they affect your results in this experiment? How could you have avoided satellite colony formation?
  7. In the diagram of the pGLO GFP operon with its regulatory elements, why do the AraC and GFP arrows face in different directions?

References & further reading

pGLO background

pGLO Map and Resources from Bio-Rad.

Fluorescent proteins

Behind a Marine Creature’s Bright Green Fluorescent Glow. Branchiostoma (or Amphioxus), which you may remember from Bio 6A, produces its own green fluorescent protein. A press

Coral Fluorescent Proteins as Antioxidants. Palmer et al., 2009. PLOS One.

AraBAD operon

AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. Schleif, 2010, BFEMS Microbiology Reviews. A detailed review of the mechanisms of the operon, written by the guy who figured it out.

Regulation of the L-arabinose operon in Escherichia coli. Schleif.

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