Pseudomonas and Streptomyces Microbiology Techniques

Pseudomonas and Streptomyces Microbiology Techniques

The handout details microbiological techniques for isolating genomic DNA from Pseudomonas and Streptomyces species. It emphasizes the importance of glycerol as a cryoprotectant for bacterial cultures stored at -80°C. The document outlines procedures for DNA purification using a DNeasy kit, highlighting the roles of lysis buffer and Proteinase K in breaking down cellular components. Additionally, it covers agarose gel electrophoresis methods for visualizing DNA fragments, essential for confirming successful DNA isolation. This resource is ideal for microbiology students and researchers focusing on bacterial taxonomy and genetic analysis.

Key Points

  • Describes procedures for preparing glycerol stocks of Pseudomonas isolates for long-term storage.
  • Outlines the genomic DNA isolation process using a DNeasy kit tailored for Gram-negative bacteria.
  • Explains the role of Proteinase K and SDS in lysing bacterial cells during DNA extraction.
  • Details agarose gel electrophoresis techniques for visualizing DNA fragments post-isolation.
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  • Niesha Dupree
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Week 11. Pseudomonas project Day 6 and Streptomyces project Day 4
11.1 Pseudomonas project Day 6: Preparation of glycerol frozen stock and
Pseudomonas genomic DNA
Day 6 Introduction
Prokaryotic taxonomy, the study of naming, defining, and classifying groups of
biological organisms based on shared characteristics, has traditionally involved the
classification of organisms grown in pure culture on the basis of their phenotypes and
biochemical characterization (e.g., Gram status, ultrastructure, carbon source
utilization, and susceptibility to antibiotics).
In the 1970s, Carl Woese pioneered molecular taxonomy focusing on the nucleic
acid sequences of ribosomal RNA (rRNA), a molecule found in abundance in all
organisms. The most direct way to identify and classify an organism is to determine
the DNA sequence of the gene that encodes its 16S rRNA, and to compare it to the
sequences found in public databases. Those sequences, which show the highest
degree of similarity to the query sequence, are those that derive from the species
closest relatives. Bear in mind that you will isolate and amplify the DNA that encodes
the 16S rRNA. As these two molecules have complementary sequences, you can
deduce the RNA sequence by determining the DNA sequence. The results from
analyzing the rRNA sequence will reveal the species of the organism.
In today’s exercise, we will primarily focus on isolating genomic DNA from our
Pseudomonas isolates which will be used as templates to amplify the16s rRNA gene
next week. Before the isolation of genomic DNA, we first need to preserve our
Pseudomonas isolates as glycerol frozen stocks at -80°C.
Day 6 Procedure: Preparation of glycerol frozen stock for storage of your
Pseudomonas isolate
Glycerol stocks of bacterial strains can be stored indefinitely at -80°C. Prior to
freezing it is important to add glycerol, a cryo-protectant, to the cultures. Glycerol
stocks are easily prepared by mixing liquid culture with glycerol in screwcap tubes.
Later inoculating fresh media with a small amount of the frozen stock can revive the
bacterial strains.
Materials (each group)
• 2 TSB broth cultures of Pseudomonas isolate, each student picks their own
isolate
• 1 tube of sterile glycerol (50%, v/v)
• 2 screwcap tubes
• Sterile micropipette tips (1 box of 1000 µL and 1 box of 200 µL)
Note: While freezing down your Pseudomonas isolate, make sure you work near the
flame to avoid contamination.
Procedure
• Calculate how much glycerol 50% (v/v) should be mixed with your culture to
get 1 ml of a suspension of cells in 15% (v/v) glycerol.
_____ µl of culture + _____ µl of 50% glycerol
• Label a screwcap tube with your designated number on the cap as well as on
the side using a sharpie marker.
• Add calculated amount of culture and 50% glycerol into the screwcap tube and
mix it by vortexing at low speed.
• Leave the screwcap tube in the designated box on the front bench.
• Fill the required information in the spreadsheet beside the box.
Preparation of genomic DNA from Pseudomonas isolates
The bacterial genome, all the hereditary material (DNA) contained within a given cell,
is comprised of both the chromosome and any plasmid DNA that may be present in
the cytoplasm. The size of a bacterial genome varies from species to species, but
ranges from about 1.6 Mb (megabase pairs, or 1.6 X10
6
base pairs) to about 10 Mb.
Free-living microbes with greater metabolic versatility, such as Pseudomonas and
Streptomyces, tend to have larger genomes than obligate intracellular parasites, such
as Mycoplasma and Rickettsia.
You will isolate and purify genomic DNA from your Pseudomonas isolate as the initial
step in determining its 16S rRNA sequence. During the isolation process, the DNA
molecule will be broken into pieces of about 20-30 kb (kilobase pairs, or
20,000-30,000 bp). As plasmids are generally much smaller, they will remain intact
throughout the purification process.
A DNeasy kit (produced by Qiagen) specifically designed for genomic DNA
purification from Gram-negative bacteria will be used. It is important to fully
understand each step of the purification process and the chemical nature and
function of the solutions provided in the kit. It is a part of this exercise to understand
and describe how each of the solutions acts to purify genomic DNA away from the
other cellular components.
Summary of the key steps of DNA purification:
• The bacterial culture is centrifuged to separate the cells from the medium in which
they were growing, and the spent medium is discarded.
• Lysis buffer, which contains sodium dodecyl sulfate (SDS) is added to solubilize
membranes.
• The addition of Proteinase K, an enzyme that hydrolyzes most proteins (except,
of course, itself). This enzymatic reaction is performed at 56°C, which is the optimal
temperature for this enzyme.
Note: Given the structure of the Gram-negative cell wall (an outer membrane, a thin
layer of peptidoglycan, and an inner membrane), the combination of SDS and
Proteinase K will solubilize both membranes, and hydrolyze the peptide bonds found
in the peptidoglycans. Proteinase K will also hydrolyze most of the intracellular
proteins once the cells have been lysed. Recall that enzymes can perform many
cycles of their catalytic activity, and allowing the reaction to proceed for 20 minutes
ensures that virtually all proteins in the mixture are hydrolyzed.
• The addition of ethanol to the mixture causes the DNA to precipitate from
solution. This causes a physical change, in which the ethanol dehydrates the DNA.
The individual DNA molecules have a greater affinity for one another than they do for
that solvent, and the result is precipitation from solution.
• The entire mixture of precipitated DNA is then applied to the DNeasy mini column.
Only DNA binds to the column, other components (e.g., proteinase K) pass through
the column, resulting in isolation of the DNA.
• The remaining washing steps with ethanol-based buffers (AW1 and AW2) serve
to wash off remaining non-DNA molecules. In each case, the DNA remains bound to
the column matrix and contaminants are washed into the collection tube.
Centrifugation provides the force to wash the solutions over the bound DNA.
• The final step is elution of the DNA from the column matrix, which is done by
adding an aqueous elution buffer (AE) to the column with the bound DNA. It is
important to remember to place the column into a clean microcentrifuge tube for this
final centrifugation step, since the wash-through will contain the purified soluble DNA.
Agarose Gel Electrophoresis
Note: This part will be done by the instructor, but you need to understand the
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FAQs of Pseudomonas and Streptomyces Microbiology Techniques

What is the role of glycerol in bacterial culture preservation?
Glycerol acts as a cryoprotectant when added to bacterial cultures before freezing. It helps prevent the formation of ice crystals that can damage cell membranes during the freezing process. By maintaining the integrity of the cells, glycerol allows for the long-term storage of bacterial strains at -80°C. This method ensures that viable cultures can be revived later for experimental use.
How is genomic DNA isolated from Pseudomonas species?
Genomic DNA isolation from Pseudomonas involves several key steps. Initially, bacterial cells are harvested by centrifugation, followed by resuspension in PBS buffer. A lysis buffer containing SDS and Proteinase K is added to break down cell membranes and proteins. Ethanol is then introduced to precipitate the DNA, which is subsequently purified using a DNeasy spin column. This process results in high-quality genomic DNA suitable for further analysis.
What is the significance of agarose gel electrophoresis in DNA analysis?
Agarose gel electrophoresis is crucial for separating and visualizing DNA fragments based on size. During this process, DNA, which is negatively charged, migrates towards the positive electrode when an electric field is applied. The speed at which DNA fragments move through the gel is inversely proportional to their size, allowing for effective size estimation. This technique is essential for confirming the successful isolation of genomic DNA and for analyzing PCR products.
What are the key components of the DNeasy kit used for DNA purification?
The DNeasy kit contains several critical components for DNA purification, including lysis buffer, Proteinase K, and ethanol. The lysis buffer, which contains SDS, disrupts cell membranes, while Proteinase K digests proteins, facilitating the release of DNA. Ethanol is then added to precipitate the DNA from the solution. The kit also includes spin columns that selectively bind DNA, allowing for the removal of contaminants and yielding purified genomic DNA.
What techniques are used to check for contamination in Streptomyces cultures?
To check for contamination in Streptomyces cultures, researchers typically re-streak colonies onto fresh agar plates. This method allows for the isolation of pure colonies by transferring a small amount of the original culture onto a new plate. Observing colony morphology and growth patterns helps identify potential contaminants. Incubation at an appropriate temperature, such as 30°C, further supports the growth of Streptomyces while inhibiting unwanted microbial growth.
What factors influence the migration of DNA during agarose gel electrophoresis?
The migration of DNA during agarose gel electrophoresis is influenced primarily by the size of the DNA fragments and the gel's concentration. Smaller DNA fragments migrate faster through the gel matrix, while larger fragments move more slowly. Additionally, the overall charge of the DNA, which is negative due to phosphate groups, drives its movement towards the positive electrode. These factors are critical for achieving accurate separation and visualization of DNA samples.
Why is it important to include molecular weight markers in gel electrophoresis?
Including molecular weight markers in gel electrophoresis is essential for estimating the sizes of DNA fragments in the samples. These markers consist of DNA fragments of known sizes, providing a reference for comparison. By running these alongside experimental samples, researchers can accurately determine the size of their DNA fragments based on their migration distance relative to the markers. This practice enhances the reliability of the results and aids in the interpretation of DNA analysis.

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