CHAPTER 5 BACTERIAL TRANSFORMATION & POLYMERASE CHAIN REACTION

What is the clinical significance of Bacterial Transformation in biotechnology?

Antibiotic resistance spread enables genetic engineering for insulin production, and underlies gene therapy vectors.

When did Griffith discover transformation?

1928

How did the mouse die in station 1?

From pneumonia in a live s-strain virus

What did the S-Strain contain?

Polysaccharide capsule

How was the R-Strain different than the S-Strain?

Lacks a capsule

What happened in Station-3 of Griffith’s experiment?

Mouse survives— dead bacteria cannot replicate

What happened in Station-4?

Heat-killed s strains and a mix of r strain kills the mouse

How did the bacteria demonstrate genetic variation?

R-strain acquired circulene from dead S-strain DNA

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

x

What happens during DNA integration?

x

What happens during bacterial division in the transformation process?

x

What is the clinical significance of Bacterial Conjugation?

x

What are the different bacterial mating types?

x

What is the Fertility factor?

x

What is the Conjugation Bridge?

x

How are genes transferred during Conjugation?

x

What is high frequency Recombination?

x

What is Bacterial Transduction?

x

What happens during Bacteriophage attachment?

x

What happens during phage DNA replication?

x

What happens during packaging and phage release?

x

What are the steps of generalized transduction?

x

What happens during prophage integration and excision?

x

What is hybrid DNA formation?

x

How is DNA transferred to recipient?

x

What is the pre-integration state?

x

What is the post-integration state?

x

What is the clinical significance of Bacterial Transduction?

x

How did genetic engineering revolutionize medicine?

What is the Donor Plasmid?

Small, Circular DNA molecule isolated from E. coli bacteria. Unlike the bacterial chromosome, plasmids are easily manipulated ringlets of DNA that serve as vectors or genetic engineering.

What are Restriction enzymes?

Molecular scissors that cleave DNA at specific recognition sequences. Creates sticky ends that allow precise DNA fragment insertion. Discovered in the 1960s, these enzymes opened the door to modern recombinant DNA technology.

What are DNA ligase?

Molecular glue that seals DNA strands together. Operates during DNA replication and repair by forming phosphodiester enzymes between nucleotides are the restriction sites.

Where do foreign DNA come from?

Various sources including other bacteria, animal tissues (mouse), or human cells.

What is a chimera?

The recombinant plasmid created when foreign DNA combines with the donor plasmid at the restriction point. This hybrid molecule (mythological lion-goat-serpent monster) now carries genetic instructions from a different organism.

What does CaCl2 solution do to cells?

• Opens cell wall and membranes

• Permits chimera entry into bacterial cytoplasm

• Allows cooling and membrane resealing

How does the host bacteria reproduce quickly?

Through binary fission

How can recombinant proteins be used?

For medical use

How did PCR revolutionize medicine?

By enabling rapid Covid-19 testing, cancer mutation screening, forensic DNA analysis paternity testing, and detection of infectious diseases from tiny samples.

What are primers?

Two short single-stranded DNA oligonucleotides

What are dNTPs?

The building blocks for new DNA synthesis

What is Taq DNA polymerase?

An enzyme that synthesizes new DNA strands

What are the three steps of PCR?

1. Initialization/Denaturation

2. Annealing

3. Elongation

What temp does denaturation occur at?

95C

What happens during the denaturation phase?

Sample is heated to approximately 95C

What happens during annealing phase?

Temperature is lowered to approximately 50-65C

What temp does annealing happen?

55C

What temp does elongation happen at?

72C

What occurs during elongation stage?

Temperature is raised to 72C (optimal for Taq polymerase activity)

How does PCR multiply? (mathematical formula)

2^n

How many cycles of PCR are typical?

25-40 cycles