Bacterial strains & plasmids

 Background

You will use a variety of different genetic types, or strains, of E. coli bacteria in the Bio 6B lab. You'll learn to culture them in several different ways, either to produce cells as a source of DNA and protein, or to answer experimental questions. Once you have the cells, you will use a range of DNA and protein techniques to investigate their specific genetic characteristics.

A single bacterial species such as Escherichia coli can encompass a wide range of genetic variation. Different strains of E. coli can be very different (such as the beneficial strains living in your intestine compared to the pathogenic strains that can make you very sick), or may differ only with respect to a single gene. In 6B, you'll work with bacterial strains that differ both in terms of their chromosomal DNA and their plasmid DNA.

Chromosomes and plasmids

Many (but not all) species of bacteria contain a single chromosome per cell. Like eukaryotic chromosomes, a bacterial chromosome consists of a DNA double helix with some protein bound to it. Bacterial chromosomes are different from those of eukaryotes in two important ways. First, bacterial chromosomes commonly form a large ring, or circular DNA molecule (eukaryotic chromosomes are linear). Second, bacterial chromosomes don't have histone proteins, while eukaryotic chromosomes have numerous histones attached to the DNA. Eukaryotic chromosomes contain much more protein overall.

The chromosome of E. coli contains about 4.6 x 10⁶ base pairs of DNA, or 4,600 kb. For comparison, the complete haploid human genome is 3 x 10⁹ base pairs.

In addition to the single chromosome found in bacteria such as E. coli, there can also be one or more plasmids. Like a chromosome, a plasmid is a circular, usually double-stranded DNA molecule. However, plasmids are much smaller; in Bio 6B, you'll use plasmids that are around 5,000 base pairs long (5 kb). Plasmids typically contain a few protein-coding genes. These genes are not part of the core genome of the species, but may be essential for survival in particular environments. For example, you'll work with plasmids that carry genes for antibiotic resistance. Plasmids play a vital role in the rapid evolution of bacteria.

Not every bacterial cell has a plasmid, but every cell must have at least one chromosome.

Plasmids can be transferred by conjugation

Bacterial conjugation is the transfer of a copy of a plasmid from one bacterial cell to another.

When two E. coli cells conjugate, the plasmid donor cell and the plasmid recipient cell must first physically attach to each other. Once this attachment is made, a translocation channel helps connect the two cells. The plasmid donor cell separates the two strands of the plasmid's double helix, and a single-stranded copy of the plasmid DNA is copied from the donor to the recipient. Each cell then uses the single strand as a template to produce a double-stranded plasmid molecule. The end result is that the recipient gains a new plasmid, but the donor still has a copy of its plasmid.

Conjugation requires a specific set of proteins; the genes for some of these proteins are on the plasmid itself, so cells that acquire a new plasmid through conjugation can then pass on a copy to another cell through conjugation. Not all plasmids contain the genes necessary for conjugation. In the 6B lab, the plasmid pARO has the genes needed for conjugation, but pGLO does not.

Conjugation is a mechanism of horizontal gene transfer. In horizontal gene transfer (also called lateral gene transfer), DNA is passed from one cell to another, already existing cell. (This is in contrast to vertical gene transfer, which is when genes are passed from parents to their offspring.) Plasmids are frequently passed from one bacterial cell to another, and this allows genes contained on plasmids to be shared widely among different cells. This is one of the reasons bacteria can evolve so fast: individual cells can acquire new genes from other cells, even if those other cells are not the same species.

Bacterial cells can also acquire plasmids through transformation

In the process of transformation, bacteria take up plasmid (or other) DNA directly from the environment. Since DNA molecules are charged and very large, it's not easy for them to cross cell membranes. In natural environments, transformation sometimes happens as a controlled process involving specific protein channels for taking up the DNA. Thus, transformation is also an important mechanism of horizontal gene transfer. In the 6B lab, you will use some trickery to transform E. coli with the plasmid pGLO, which will then allow you to study the genes contained on the plasmid.

The transformation process is inefficient; most of the cells in your sample won't end up being transformed with the plasmid. To get a pure culture of bacteria containing the plasmid, you'll need to weed out the non-transformed cells using antibiotics to create a selective medium, as described below.

Plasmids as molecular biology tools

Gene cloning of the fundamental approaches of molecular biology. Cloning a gene means making many copies of the DNA sequence containing the gene, and this has traditionally been accomplished by inserting the DNA into a cell.

If you want to insert a gene into a bacterial cell, you need to ensure that the DNA will be copied when the cell replicates itself. For this, you need a cloning vector, which is a piece of DNA that can carry some other bit of DNA into a cell and allow the new DNA to be replicated within the cell. Plasmids are the most widely used cloning vectors in molecular biology. A cloned gene that has been inserted into a plasmid can be transferred from one cell to another, replicated within the cell, and used to study the function of the gene. Plasmid cloning vectors often contain mechanisms to allow the cloned gene to be expressed (so that a protein based on the gene is produced by the cell) and also contain an antibiotic resistance gene so that a selective medium can be used to select for the cells that contain the plasmid.

Antibiotics and antibiotic resistance

Antibiotics are chemicals that kill bacteria or prevent them from growing. Most antibiotics work by blocking enzymes or biochemical processes that are specific to prokaryotes. Some bacteria are resistant to specific antibiotics because they can make a protein that destroys the antibiotic or prevents it from acting on the cell. Such proteins are encoded by antibiotic resistance genes.

Antibiotic resistance genes are often carried on plasmids, with the result that these genes can be copied from cell to cell by conjugation or introduced into a cell by transformation. You’ll use both of these mechanisms in Bio 6B. Antibiotics will allow you to create a selective medium: a growth environment that selects for certain strains of bacteria and prevents others from growing. You’ll use two kinds of antibiotics, and strains of bacteria that are resistant to these antibiotics:

• Streptomycin (strep) inhibits bacterial protein synthesis by binding to a component of the ribosome. The bacteria are thus prevented from growing, and may eventually die. Streptomycin resistance can come from several different mechanisms, but most commonly the resistant cells have a different version of a gene for a ribosomal subunit. Streptomycin-resistant cells tolerate streptomycin and grow on strep plates because strep doesn't inhibit their ribosomes.
• Ampicillin (amp) inhibits a bacterial enzyme called transpeptidase, which is needed for cell wall synthesis. With this enzyme inhibited, the bacteria can’t make a complete cell wall, and they eventually get lysed (burst open). Ampicillin resistance is provided by the gene for β-lactamase (abbreviated bla, but commonly just called the ampicillin resistance gene). This enzyme breaks down ampicillin, allowing the bacteria to survive and grow. Thus, amp-resistant cells have an extra gene that their non-resistant relatives lack. This extra gene is commonly carried on a plasmid. In our experiments, the fact that amp-resistant bacteria secrete the enzyme that destroys amp can cause a problem: if amp-resistant colonies destroy all the amp, then colonies that are not amp resistant may also grow on the same plate. Non-amp-resistant colonies that grow surrounding amp-resistant colonies are called satellite colonies; see the pGLO transformation page for a photo.

You’ll make use of antibiotics and resistance with a two-part procedure: first, induce cells to take up a plasmid that contains an ampicillin-resistance gene, then culture the cells in a selective medium that contains ampicillin to weed out any cells that didn’t take up the plasmid. This way, you’ll end up with a pure culture of plasmid-containing cells. For more on culturing bacteria, see Starting bacterial cultures.

Review

Terms, concepts, and techniques:

• Antibiotics and antibiotic resistance
• Chromosomes vs. plasmids
• Cloning vector
• Conjugation
• Horizontal gene transfer
• Plasmids and their role in antibiotic resistance
• Satellite colonies
• Selective media
• Strain
• Transformation

Review questions

1. Why do we often get satellite colonies on ampicillin plates, but not on streptomycin plates?
2. What are the two mechanisms of horizontal gene transfer that you will investigate in Bio 6B?
3. If you transform cells with pGLO, how will you know if it worked?
4. If you want to clone a gene from an animal and insert the gene into bacterial cells so the bacterial produce the animal protein, why do you need a cloning vector?

References

Videos

What is a Plasmid? - Plasmids 101. From Addgene. Good description of plasmids and how they are used in molecular biology.

Reading

The Ins and Outs of DNA Transfer in Bacteria. (Alternate URL) Inês Chen, Peter J. Christie, David Dubnau, 2005. Science 310:1456-1460. Detailed review of the mechanisms of both conjugation and transformation in bacteria.

Plasmids in Barron's Medical Microbiology (an old version of the textbook).

Chromosomes in Bacteria: Are they all single and circular? MicrobeWiki. It's common for bacteria to have a single, circular chromosome per cell, but some species have multiple chromosomes or linear chromosomes.

What’s The Problem With Ampicillin Selection? from BiteSize Bio & NEB. Answer: Satellite colonies.