studying DNA using polymerase chain reaction (PCR)

Background

The regulation of bacterial gene expression has evolved as an evolutionary call and
response to ever-changing environmental stimuli, particularly for those organisms with a
broad host range. The ability of bacteria to sense pH, temperature, oxygen gradient,
nutrient availability, cell density, etc. mediates coordinated outcomes including antibiotic
resistance and the expression of capsules, structural polysaccharides, secretion
systems, toxins, appendages for motility and adherence, biofilm-associated extracellular
matrix components, etc. The lists of fitness benefits are expansive and, like the
mechanisms themselves, evolving thanks in large part to the elucidation of novel
virulence pathways by modern molecular tools such as DNA and RNA sequencing,
proteomics, metabolomics, and bioinformatics software.

Identifying bacteria to the species level only provides a glimpse into the roles of that
microbe in the environment and its potential to affect animal or human health. Horizontal
gene transfer, mutation, and mechanisms of genomic rearrangement can all contribute
to different phenotypes even amongst bacteria of the same species. Therefore, while
you used biochemical tests, cell morphology, and staining techniques to identify your
ARI’s genus and species, there is still more to uncover in the genome.

Historically, researchers have been unable to characterize functions for the great
majority of bacteria inhabiting planetary soils and mammalian tissue because the great
majority of these microbes do not grow on classical laboratory media. Using a novel co-
culture model, new studies show that certain intestinal bacteria require eukaryotic cells
to provide a scaffold for in vitro growth. Biotechnology companies have further
circumvented this problem by manufacturing kits that extract and purify plasmid or
genomic DNA from bacteria found in environmental or clinical samples including blood,
saliva, and stool. Procedural steps in a typical kit include: (1) suspension and lysing of
bacterial cells using a combination of lysozyme and proteinase K; (2) stabilization of DNA
and removal of cellular debris using a series of salt buffers; (3) binding of nucleic acids
to a column containing a porous micro-filter; (4) washing of DNA by ethanol and (5)
elution of DNA using a vacuum manifold or centrifuge. Isolation of nucleic acids using
molecular kits allows scientists to identify microbes that defy lab cultivation and provides
a critical first step toward elucidating their roles in health and disease.

A critical step in studying DNA is to amplify small copy numbers through a polymerase
chain reaction (PCR). As a brief review, double-stranded

DNA is denatured in each
round of PCR and the liberated single strands serve as templates for primer annealing
and subsequent amplification. Thus, one copy of a target gene amplifies to 68 billion by
the 35th cycle (235 = 6.8 X 10 10  copies).

Lab overview

In this lab, you will continue to develop your scientific methods skills by identifying a
gene that may be present in your ARI and that can be found via a PCR-based
experiment. That means, you will have to go through the process of asking a relevant

question, creating a hypothesis, and finding information through researching the
literature. Time to put on your science hats and get started!

Instructions

1. Identify your ARIs

You will first need to identify your American River Isolate (ARI) to the genus and species
level. This can be worked on during lab time individually and with help from your team
and instructor. The remainder of the steps should be done individually for this @Home
activity.

2. Ask a question

What do you want to know about your isolate? Do you want to know how it responds to
acid stress? Or whether the isolate encodes toxins or other virulence genes? Or what
about antibiotic resistance genes? Or if it can grow in ice cold temperatures. There are
many questions you can ask about your isolate.

3. Read the literature and determine what gene you want to search for in your
ARI

Once you know the genus and species of your ARI, start reading the published literature
to get to know more about your organism and to identify a gene of interest. Your gene
must be one that could be found in the genus or species that your ARI is in (for
example, if your ARI is Pseudomonas syringae, do not look for a gene that is known to
only be present in Escherichia coli). The gene should also not be one that is highly
conserved across several bacterial lineages. This means avoid genes that are involved
in central carbon metabolism, common housekeeping genes, 16s RNA, etc.

The gene must also tell you something about the role of your microbe in the
environment, how it responds to changes in the environment, or the potential to affect
animal or human health. Some examples of these could include metabolic genes for
unique or secondary metabolic pathways, virulence genes, antibiotic resistance genes,
and stress response genes. (Note: The 16s rRNA gene does not fit this criterion and
thus should not be used for this project.)

4. Answer the questions below, and upload your answers and
references to CANVAS.

a. What is the question you are asking regarding this experiment?

b. What is your gene of interest and why did you select it? Your response
should include details on what the gene product does, and the relevance of
that to an isolate found in the American River.

c. Based on what you know about your isolate, where it was found, and
information from the literature, do you expect to find your gene of interest in
your isolate (this is your hypothesis)? Why or why not?

d. How would a PCR method help you answer your question and test your
hypothesis? Your response should include a description of what results you
would expect to find if you performed a PCR experiment .