Lecture Notes on Protoplast Fusion, Bacterial Genomic DNA Isolation, and PCR

Flashcards for reviewing key concepts and procedures discussed in the lectures.

What is a protoplast?

A cell without a cell wall; it is a bacterial, fungal, or plant cell that has had its cell wall completely or partially removed using either enzymatic or mechanical means.

Which enzymes are used to remove the cell wall from plant cells to produce protoplasts?

Cellulase, pectinase

Which enzyme is used to remove the cell wall from bacterial cells to produce protoplasts?

Lysozyme

Which enzymes are used to remove the cell wall from fungal cells to produce protoplasts?

Chitinase, glucanase

Which enzymes are used to remove the cell wall from Streptomyces cells to produce protoplasts?

Lysozyme, achromopeptidase

What is protoplast fusion?

A physical process where two or more protoplasts interact in the presence of fusion-inducing substances, resulting in hybrid products capable of dividing and regenerating cell walls.

What barriers can be bypassed by protoplast fusion?

mating types and sexually incompatibility group

What are heterokaryons?

Cells that contain genetically unlike nuclei.

What are the classifications of protoplast fusion?

Spontaneous fusion, induced fusion (mechanical fusion, chemofusion, electrofusion)

What is mechanical fusion?

Isolated protoplasts are fused mechanically under the microscope using a micromanipulator and perfusion micropipette, requiring physical contact.

What is electrofusion?

Mild electric fields in the protoplast suspension induce fusion and is more efficient than chemical fusion.

What are the two types of current used in electrofusion?

Alternating Current (AC) and Direct Current (DC)

What is chemofusion?

Fusion of freely isolated protoplasts from different sources with the help of fusogens.

What is yeast minimal medium with KCl used for?

A medium that allows selection for regenerants that complement each other's auxotrophies.

What is Tris-Sorbitol Buffer Solution used for?

A solution that provides an osmotic stabilizing environment that prevents protoplasts from lysing due to osmotic shock.

What is 0.2% of 2-mercaptoethanol in 50 mM EDTA used for?

To weaken the yeast cell wall by reducing disulfide bonds and chelating divalent cations.

What is Enzyme Solution (Zymolyase) used for?

Used to degrade the yeast cell wall, particularly β-1,3-glucan, enabling protoplast formation.

What is PEG-CaCl2-TS Buffer Solution used for?

Promotes controlled fusion of protoplasts by creating a chemically and osmotically suitable environment.

What is Malt extract yeast extract glucose peptone agar and broth (MYGP) used for?

Suitable for the cultivation, isolation, and maintenance of yeasts, molds, and other aciduric microorganisms.

What is detergent used for in protoplast isolation?

Used to disrupt cell membranes to induce protoplast bursting.

What is m2a medium with KCl used for?

Used to support the growth of both protoplasts and non-protoplasts by maintaining a balanced osmotic environment.

What is m2b medium without KCl used for?

Creates a hypotonic environment that leads to the lysis of protoplasts, thereby permitting only non-protoplasts to survive and proliferate.

What is the formula for calculating protoplasting efficiency?

CFU/mL on M2A - CFU/mL on M2B, divided by CFU/mL on M2A multiplied by one hundred.

How to calculate cell concentration when using direct microscopic count (hemacytometer) for protoplast isolation?

Cells per mL = ave. counts x CF x DF, where CF = 2.5x10^5 (for yeast only)

How to count budding cells during direct microscopic count (hemacytometer)?

If 50% or greater count separately, if less than 50% count mother cell only.

How does Fluorescein Diacetate (FDA) help in detecting protoplasts?

Live protoplasts can convert FDA into fluorescent compounds, which are then visualized under a fluorescence microscope.

How does Calcofluor White (CFW) help in detecting protoplasts?

Helps determine the onset of cell wall formation in protoplasts and is useful for assessing viability.

How does Evans Blue help in detecting protoplasts?

A non-permeating dye that is excluded by the cell membrane, indicating cell viability.

Which tests can assess DNA damage in protoplasts?

Gel electrophoresis with ethidium bromide staining and the Comet assay

What is the formula of calculating efficiency of fusion?

CFU/mL of regenerants in Yeast Minimal Medium with KCl divided by CFU/mL of regenerants on M2A multiplied by 100.

How are recombinants obtained during protoplast fusion? First step.

Both parent strains had their cell walls enzymatically removed, which allows fusion.

How are recombinants obtained during protoplast fusion? Second step.

After centrifugation, the protoplasts were resuspended in PEG-CaCl - TS buffer solution wherein PEG induces membrane fusion and calcium ions stabilize the interaction.

How are recombinants obtained during protoplast fusion? Forth step.

Plating on Yeast Minimal Medium with KCl selects for recombinant cells Note that the recombinants are now PROTOTROPHS after fusion, which means they can grow on minimal medium.

What is bacterial genomic DNA isolation?

Extracting high-quality, pure genomic DNA and separating it from other cellular components for downstream applications.

What are the advantages of CETYLTRIMETHYLAMMONIUM BROMIDE (CTAB)– BASED METHOD?

High yields, high-quality DNA, cost-effective, time-efficient

What are the disadvantages of CETYLTRIMETHYLAMMONIUM BROMIDE (CTAB)– BASED METHOD?

Time-consuming, hazardous chemicals, contamination (if not handled carefully), manual separation

What is the size and G-C content of Escherichia Coli ATCC 25922?

5.20Mb or 5, 200, 000 bp and 50.4% G-C content

What is Luria-Bertani (LB) broth used for?

General purpose medium commonly used in molecular biology procedures as pre-culture media to prepare bacteria for downstream purposes

What is the role of CTAB (cetyl trimethylammonium bromide) in DNA extraction?

To lyse cells and separate DNA from other cellular components.

How do you prepare agarose gel?

Prepare 0.8% agarose gel by mixing 0.48g of agarose in 35 mL 0.5X TAE buffer

At which step do you visualize DNA bands with UV during Gel Electrophoresis?

Visualize DNA bands with UV Illuminator and take photograph of the gel

What does faint but distinct bands around ~1645 bp on a gel indicate?

Likely the result of partial degradation of genomic DNA

Why genomic DNA is too large to resolve clearly as a single band?

Appears as high-molecular-weight band, not full size. DNA folds, coils, or shears during isolation, resulting in high-molecular- weight band near12,000 bp

How can degradation caused by genomic DNA isolation be avoided?

Keep samples cold during extraction; avoid excessive mechanical stress/shearing

What does A260/A280 ratios below < 1.8 indicate about the quality of isolated DNA?

Protein contamination

What does A260/A280 ratios below > 2.0 indicate about the quality of isolated DNA?

RNA contamination

What additional step(s) must be performed to get a A260/A280 ratio of 1.8 if less than 1.8?

re-extract the DNA using a phenol-chloroform method

What additional step(s) must be performed to get a A260/A280 ratio of 1.8 if greater than 2.0?

RNAse Treatment

What is Polymerase Chain Reaction?

A molecular biology technique used to amplify specific DNA sequences in vitro.

What happens during pre-PCR Initial Denaturation?

Heats the reaction to separate double-stranded DNA completely before cycling begins.

List the four steps of Polymerase Chain Reaction.

Denaturation, Annealing, Extension, Final Elongation

What is the equivalent cellular DNA replication strand separation of the step PCR Denaturation?

Done by helicase, an enzyme that unwinds the DNA

What type of primer is used in PCR?

Uses synthetic DNA primers designed for target specificity

What DNA Synthesizing Enzyme is used for PCR?

Uses Taq polymerase, which is heat-stable but lacks proofreading

What temperature condition is needed for PCR?

Requires cycling temperatures (denaturation, annealing, extension)

What is the method for Conventional PCR?

The standard method involving denaturation, annealing, and extension steps in a thermocycler to amplify DNA

What is 16S Ribosomal RNA Gene?

A highly conserved component of the prokaryotic 30S small ribosomal subunit.

What is Gel Electrophoresis?

A laboratory technique used to separate DNA fragments based on their size by applying an electric current through an agarose gel.

What is the function of PCR buffer?

Maintain optimal pH and ionic strength for Taq polymerase activity.

What is the function of MgCl2 in PCR?

Facilitates primer-template binding and activates DNA polymerase; cofactor for the enzyme.

What are primers?

Short, single-stranded oligonucleotides designed to be complementary to specific sequences on the target DNA.

What is the dNTP mix made of?

Supplies nucleotides (dATP, dTTP, dCTP, dGTP) for the elongation of new DNA strands.

What is Taq Polymerase?

A heat-stable enzyme that catalyzes the synthesis of new DNA strands.

What is Nuclease Free Water?

Used to adjust volume and prevent degradation of nucleic acids.

What happens during PCR Denaturation?

Double-stranded DNA (dsDNA) is heated to around 94°C

What happens during PCR Annealing?

Temperature is lowered to approximately 55°C. Allowing short primers to bind (anneal) to their complementary sequences on the single-stranded DNA

What happens during PCR Amplification?

Temperature is raised to about 72°C, the optimal condition for Taq polymerase

Why is PCR normally carried out for about 30 cycles?

Each cycle ideally doubles the amount of DNA. After 30 cycles, this results in over 1 billion copies (2 ) from a single DNA template.

What will happen if primers designed to anneal to the template DNA on either side of a target gene both add dNTPs in the same direction? Explain your answer.

Incorrect Orientation and No Overlap or Amplification since DNA polymerase can only synthesize DNA in the 5′ to 3′ direction

What does your negative control show?

PCR reagents, primers, and pipetting process were free from DNA contamination

Why is it important to wear gloves when setting up a PCR reaction?

Prevents DNA Contamination

What are the application of PCR in Disease Diagnosis?

Detects pathogens like viruses (e.g., SARS-CoV-2, HIV), bacteria, or genetic mutations in clinical samples

What are the application of PCR in Genetic Fingerprinting / Forensic Analysis?

Identifies individuals using unique DNA profiles from biological evidence such as blood, hair, or saliva

What are the application of PCR in Molecular Cloning and Gene Expression Studies?

Amplifies specific genes for insertion into vectors or for measuring gene activity levels

What are the application of PCR in DNA Sequencing Preparation?

Enriches specific DNA regions before sequencing, especially in targeted or next- generation sequencing workflows.

What are the application of PCR in Detection of Genetically Modified Organisms (GMOs)?

Identifies transgenes or genetic modifications in food products or crops.