The Plasmid Labs

You'll use plasmids for many different experiments in Bio 6B. Much of your lab time will be centered on two separate, but overlapping, projects with two different plasmids: conjugation with pARO180 and transformation with pGLO. You'll use these plasmids to investigate methods for getting DNA into cells, controlling the expression of a gene on pGLO, and analyzing both DNA and protein from cells containing the plasmids. The specific methods will be described on other pages; this page is intended to give you an overview and emphasize the connections among the various experiments you'll do.

Here is an overview of the whole approach, to be carried out over multiple lab periods:

Working with bacterial cells

1: Conjugation

Conjugation is the transfer of a plasmid from one bacterial cell to another. You'll mix two strains, or genetic types, of E. coli bacteria. The S strain (actually called S-17-1) contains the plasmid pARO (actually called pARO180), which can be transferred through conjugation. The H strain (HB101) doesn't have a plasmid to begin with, but receives pARO through conjugation. The plasmid contains a gene called bla (β-lactamase), which gives the cells resistance to the antibiotic ampicillin (amp). See Conjugation.

2: Transformation

Transformation is when cells take up DNA from the environment, rather than from another cell. You'll transform H cells with the plasmid pGLO. Unlike pARO, pGLO lacks the essential genes for conjugation. However, pGLO has a gene for green fluorescent protein (GFP), which you'll be able to experiment with, and it also has the ampicillin resistance gene bla. See pGLO.

3: Plating

Plating means growing your bacterial cultures on plates, or petri dishes containing agar with specific growth media. The most important outcome will be that you can use plates with ampicillin as a selective medium: only cells containing a plasmid (either pARO or pGLO) will grow with the antibiotic, since the plasmids provide antibiotic resistance. In addition, you'll use various other plates as experimental treatments or as controls to investigate specific aspects of your experiment. These plates are described on the conjugation and pGLO pages. See Conjugation and pGLO.

All the steps described so far (conjugation, transformation, and plating) use classical microbiology techniques. You genetically manipulate your cells, then infer the outcome based on the characteristics of the bacterial colonies you observe. To bring these experiments into the realm of molecular biology, the next steps would be to directly examine the DNA and proteins from the cells, using the steps described below.

Working with DNA & protein

4: Total nucleic acid gel

If conjugation is successful, your conjugation plate results tell you that a new double-resistant bacterial strain (growing on amp & strep together) was generated when you mixed two different single-resistant strains (growing on either amp or strep alone). From this, you could reasonably infer that plasmid DNA was transferred from one cell to another in conjugation. You can verify this directly by purifying the DNA from all three strains and examining it on an electrophoresis gel.

You'll extract DNA and RNA from bacterial colonies on your conjugation plates, and analyze these nucleic acids on an electrophoresis gel. You should be able to see both chromosomal DNA (present in all cells) and plasmid DNA (present in some cells). This should allow you to verify that the H strain does not have plasmid DNA before conjugation, but it does have a plasmid after conjugation. The chromosomal DNA should look the same for all the samples, and serves as a control for the efficacy of DNA extraction.

You'll perform these steps only with bacterial colonies from the conjugation experiment. See Total Nucleic Acid Extraction for the extraction technique, and DNA Electrophoresis Method for the gel.

5: Purify plasmid; restriction digest & gel

At this point, you should have colonies with pARO and colonies with pGLO. You can verify that the cells contain the expected plasmid by examining the plasmid DNA through electrophoresis. First, you'll perform plasmid purification, eliminating the chromosomal DNA and the RNA. The purified DNA of the two plasmids will look about the same on a gel, because the pARO and pGLO plasmids are fairly close to the same size. You'll use restriction enzymes to cut the plasmid DNA. Since the two plasmids have different nucleotide sequences, and restriction enzymes cut DNA at specific nucleotide sequences, the plasmids will be cut at different locations and will produce DNA fragments of different sizes. You'll be able to analyze the DNA sequences to predict the exact results of the cutting using bioinformatics software, then verify your results on a gel.

You'll perform these steps using colonies from both the conjugation (pARO) and transformation (pGLO) plates. See Plasmid Purification, Virtual Digests, Restriction Digests, and DNA Electrophoresis.

6: Analyze proteins: chromatography & electrophoresis

The plasmid pGLO contains an operon that encodes green fluorescent protein. Once you've transformed E. coli cells with pGLO, you'll be able to induce the cells to produce large quantities of GFP; the cells will then be brightly fluorescent when viewed under UV light. You'll know that GFP is present when you see the green fluorescence.

To learn more about the cells' complete set of proteins, you can then extract the proteins from the cells and analyze them using hydrophobic interaction chromatography (not shown in this diagram) and protein electrophoresis. The gel image shows that the cells always contain numerous proteins, but GFP is present only when the expression of the GFP operon is induced by adding arabinose to the culture. See Hydrophobic Interaction Chromatography and SDS-PAGE.