CHAPTER 5 BACTERIAL TRANSFORMATION & POLYMERASE CHAIN REACTION; Tabitha Spells

What is the clinical significance of bacterial transformation in biotechnology in biotechnology?

Creating genetically engineered bacteria for different types of therapy

When did Griffith discover transformation?

1928

How did the mouse die in station 1?

Because the S-strain bacteria had a capsule enabling immune evasion

What did the S-Strain contain?

pneumonia

How was the R-strain different from the S-strain?

presence of the polysaccharide capsule in the S-strain, which the R-strain lacked, enabling the S-strain to cause pneumonia. 

What happened in station-3 of griffith’s experiment?

heat killed S-Strain injected into the mouse and destroys the bacterial protein and viability, so the mouse lives

What happened in station 4?

the mouse die even though the S-strain is heat killed because some of the S-strain did not lose their capsule when being heated

How did the bacteria demonstrate genetic variation?

Because the daughter cell interied the capsule synthesis capability

What happens during DNA release and uptake in the formation process?

making the cell "competent" through chemical/electrical treatment, binding the DNA to the cell wall, fragmenting linear DNA, and using transporter proteins to move it across the membrane

What happens during DNA integration?

Competent bacteria take up foreign DNA (like plasmids) from their environment, which then either stays as an independent plasmid or integrates into the bacterial chromosome via homologous recombination, allowing the bacteria to gain new traits

What happens during Bacterial division in the transformation process?

The daughter cells replicate hybrid chromosomes containing the S-Strain casule gene

What is the clinical significance of bacterial conjugation?

its role as the major driver for the rapid spread of antimicrobial resistance genes and virulence factors among pathogenic bacteria

What are the different bacterial mating types?

F+ (plasmid only), Hfr (chromosome-linked plasmid), and F' (F plasmid carrying some chromosomal DNA)

What is the fertility factor?

a specific bacterial plasmid (F-factor) enabling gene transfer or, more broadly, to various elements impacting human reproductive success, including age, lifestyle (diet, smoking, alcohol), genetics, environment, weight, and underlying medical conditions

What is the conjugation bridge?

a temporary, protein-based channel that forms between two bacteria (a donor and recipient) during bacterial conjugation

How are genes transferred during conjugation?

through direct cell-to-cell contact

What is High Frequency Recombination?

The fertility plasmid (F-factor) has integrated into its main chromosome, making it highly efficient at transferring large chunks of its own chromosomal DNA, along with the F-factor, to other bacteria during bacterial conjugation, leading to frequent genetic recombination in the recipient cell

What is bacterial Transduction?

the process of transferring bacterial DNA from one bacterium to another via a virus (bacteriophage), acting as a genetic shuttle without direct cell-to-cell contact

What happens during Bacteriophage attachment?

The virus uses specialized structures like tail fibers to recognize and bind to specific receptors (proteins, carbohydrates) on the surface of a susceptible host bacterium, initiating the infection process, which can be reversible or irreversible and determines the phage's host range

What happens during phage DNA replication?

The virus hijacks the host bacterium's machinery to make many copies of its genome and proteins, either by directly multiplying (lytic cycle) and bursting the cell, or by integrating its DNA into the host's chromosome (lysogenic cycle) and replicating passively with the host's own DNA

What happens during packaging and phage release?

A powerful molecular motor uses ATP to force long DNA concatemers into a pre-formed, empty protein shell (prohead), compressing it tightly and cutting the DNA to size, followed by tail assembly; release then involves the tail triggering conformational changes in the head, causing the DNA to be ejected into a new host cell, often through lysis

What are the steps of generalized transduction?

Phage infection and DNA injection, bacterial DNA fragmentation, accidental packaging of host DNA into a phage head, release of transducing particles, infection of a new host, and homologous recombination of the donor DNA into the recipient's chromosome

What happens during prophage integration and excision?

Prophage excision, triggered by host stress (like DNA damage), involves integrase plus an excisionase (Xis) protein to reverse the process, cutting the prophage out to initiate the lytic cycle, often leading to cell lysis and new virus production

What is hybrid DNA formation?

The process where two complementary single strands of DNA (or DNA and RNA) bond together to form a stable double helix

How is DNA transferred to recipient?

conjugation (direct cell contact), transformation (uptake of DNA from the environment), and transduction (viral delivery)

What is the pre-integration state?

Typically refers to the Pre-Integration Complex (PIC) in retroviruses like HIV, a crucial stage where viral DNA, formed by reverse transcription, is bundled with viral and host proteins, ready to be inserted (integrated) into the host cell's genome to establish a permanent infection

What is the post integration state?

The unified, combined entity that exists after the complex process of combining two companies (or systems/teams) following a merger or acquisition, where operations, cultures, systems, and people are merged to function as a single, cohesive organization and achieve intended synergies

What is the clinical significance of bacterial transduction?

A major mechanism for the spread of antibiotic resistance genes and virulence factors among bacteria

How did Genetic engineering revolutionize medicine?

enabling the creation of targeted drugs (like insulin, Herceptin), developing gene therapies to correct genetic disorders (cystic fibrosis, blindness), powering personalized medicine through genome analysis, improving vaccines, and creating powerful diagnostic tools, all by allowing precise DNA manipulation with tools like CRISPR

What is the Donor plasmid?

a DNA molecule used as a template to deliver new genetic material or make precise edits to a genome

What are Restriction enzymes?

"molecular scissors" that cut DNA at specific recognition sequences,

What are DNA ligases?

"molecular glue," joining DNA strands by forming phosphodiester bonds

Where do foreign DNA come from?

the environment, transplants, and physical contact

What is a Chimera?

an organism containing cells from two or more genetically distinct individuals, originating from different embryos, grafts, or transplants

What does CACL2 Solution do to Cells?

makes bacterial cell membranes temporarily permeable to foreign DNA

How does host bacteria reproduce quickly?

binary fission(asexual reproduction)

How can recombinant protein be used?

for medicine (insulin, antibodies, vaccines for diabetes, cancer, infections), research (studying cell function, creating disease models), diagnostics (assays, pregnancy tests), and industry (enzymes for textiles, food processing, agriculture)

How did PCR revolutionize Medicine?

it has enabled rapid, precise DNA amplification, Changing diagnostics for infectious diseases (like COVID-19), genetic disorders (sickle cell), and cancer monitoring (minimal residual disease), and is even in cold cases/criminal cases.

What are primers?

a preparatory coating or substance that creates a base for something else, like paint or makeup, ensuring better adhesion, durability, and a smoother finish

What are dNTPS?

the fundamental building blocks of DNA, consisting of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases (Adenine, Cytosine, Guanine, Thymine)

What is Taq DNA polymerase?

Thermus aquaticus, a type of Polymerase found in bacteria from hot springs which does not denature at high temps.

What are three steps of PCR

Denaturation, Annealing, Elongation

What temp does Denaturation occur at?

80-90C

What happens during the denaturation phase?

applying heat (or chemicals/stress) to break the bonds holding a molecule's structure, causing it to unfold or separate into simpler components

What temp does Annealing happen?

45-65C

What happens during the annealing phase?

short DNA primers to bind (hybridize) to their complementary sequences on the single-stranded DNA templates, creating starting points for DNA polymerase to synthesize new DNA strands in the next extension step

What temp does elongation happen at?

72C for Taq

What occurs during the elongation stage?

synthesis of new DNA strands by DNA polymerase

How Does PCR multiply(mathematical formula)?

2^n

How many cycles of PCR are typical

30