Please read this information and Answers to the 4 questions at the end of these instructions and a bibliography that lists the literature you consulted for this project (primary research article(s), review article(s), website(s), book chapter(s), etc)
My isolate:
Genus / Species: Klebsiella pneumoniae , subspecies ozaenae
American River Isolate Genetics Assignment
Objective
Ok, so you’ve identified your isolate that was obtained from the American River. What is the next step? How would you build on this knowledge to learn more about this microbe and its importance in the ecosystem?
In this lab exercise, you will continue exploring the biology of your American River Isolate. You will perform literature research that will help you to design a PCR-based experiment. The goal is to determine whether your ARI encodes a gene that is specific to your ARI’s genus, species, or strain and would enhance the fitness of your ARI. These genes could be virulence genes, antibiotic resistance genes, stress response genes, etc.
There are two main objectives for this lab exercise:
1) To explore the literature on your identified American River Isolate.
2) To decide on “the next step” by asking a relevant question regarding the biology and genetics of your ARI, creating a hypothesis, and thinking through how you would design an experiment to address the hypothesis.
What you will submit
Answers to the questions at the end of these instructions (#4 a,b,c,d), and a bibliography that lists the literature you consulted for this project (primary research article(s), review article(s), website(s), book chapter(s), etc). All of this should be compiled into a single Word or PDF file and submitted through Canvas.
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 1010 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 .
