Amrita Rajdurai: Bacterial Transformation and Polymerase Chain Reaction

What is the clinical significance of bacterial transformation in biotechnology?

Bacterial transformation explains horizontal gene transfer mechanisms that are responsible for rapid antibiotic resistance dissemination, enabling recombinant DNA for dna tech pharmaceutical reproduction, and providing insight into genetic engineering approaches to human gene therapy

When did Griffith discover transformation?

1928

How did the mouse die in station 1?

it was injected with live virulent, smooth S, bacteria,

What did the s-strain contain?

the s-strain had a polysaccharide capsule, which surrounds the bacterial cell and causes disease, and it can be easily destroyed by immune system

How was the r-strain different from the s-strain

the r strain doesn’t have a capsule, it couldn’t cause infection, which the capsule made the s-strain deadly

what happened on station-3 of griffith’s experiment?

the mouse was injected with heat-killed s-strain bacteria. The mouse lived, no live bacteria were recovered from the mouse, so it showed that the dead-strain bacteria couldn’t cause the disease

What happened in station 4?

in station 4 the mouse was injected with live r-strain bacteria and heat killed s-strain bacteria, but the mouse STILL died, teh s-strain bacter was recovered from the mouse’s body

How did bacteria demonstrate genetic variation?

bacteria demonstrated genetic variation through transformation

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

• dead bacteria releases dna

• competent bacteria take up free dna from environment

What happens during DNA integration?

• foregin dna integrates into host chromosome

• occurs via homologous recombination

What happens during Bacterial division in the transformation process?

transformed cell divides, new rait is passed to daughter cells

What is the clinical significance of bacterial conjugation?

spreads antibiotic genes, major problem in hospitals

What are the different bacterial mating types?

f+,f-, and hfr

What is the fertility factor?

a plasmid, it has the genes for a sex pilus formation, dna transfer

What is the conjugation bridge?

A physical connection between bacteria

Allows DNA transfer

How are genes transferred during conjugation?

DNA copied and passed through pilus

One strand transferred → complementary strand made

What is high frequency recombination?

F factor integrated into chromosome

Transfers chromosomal genes efficiently

What is bacterial transduction?

Transfer of bacterial DNA via bacteriophages

What happens during bacteriophage attachment?

Phage binds to specific receptors on bacteria

what happens during phage dna replication?

Phage DNA enters cell

Host machinery makes new phage DNA and proteins

what happens during packaging and phage release?

DNA packed into capsids

Cell lyses → phages released

what are the steps of generalized transduction?

Phage infects bacteria

Host DNA fragmented

Bacterial DNA mistakenly packaged

New bacteria infected

DNA recombines

what happens during prophage integration and excision?

Phage DNA integrates into chromosome (lysogeny)

Later excised to enter lytic cycle

what is hybrid dna formation?

Donor DNA recombines with recipient DNA

how is dna transferred to recipient?

Injected by bacteriophage

what is the pre-integration state?

Donor DNA exists freely in cytoplasm

what is the post-integration state?

DNA becomes part of host genome

what is the clinical significance of bacterial transduction?

Transfers toxins and resistance genes

Example: diphtheria toxin

How did genetic engineering revolutionize medicine?

Produces insulin, vaccines, gene therapy

Enables diagnostics and personalized medicine

What is the donor plasmid?

Circular DNA carrying gene of interest

What are restriction enzymes?

Cut DNA at specific sequences

What are DNA ligase?

Join DNA fragments together

Where do foreign dna come from?

Other organisms or synthetic genes

What is a chimera?

DNA made from two different species

What does CaCl2 solution do to cells?

Makes bacterial membranes permeable to DNA

How does the host bacteria reproduce quickly?

Binary fission

Short generation time

How can recombinant proteins be used?

Medicine (insulin)

Vaccines

Industrial enzymes

How did PCR revolutionize medicine?

Rapid DNA amplification

Used in diagnostics, forensics, research

What are primers?

Short DNA sequences that start replication

What are dNTPs?

DNA building blocks (A, T, C, G)

What is taq dna polymerase?

Heat-stable enzyme from Thermus aquaticus

What are the three steps of PCR?

Denaturation

Annealing

Elongation

What temperature does denaturation occur at?

Temperature: ~94–95°C

What happens during the denaturation phase?

DNA strands separate

What temp does annealing happen?

Temperature: ~50–65°C

What happens during the annealing phase?

Primers bind to DNA

What temp does elongation happen at?

Temperature: ~72°C

What occurs during the elongation stage?

DNA polymerase extends strand

How does PCR multiply? (mathematical formula)

2ⁿ (n = number of cycles)

How many cycles of PCR are typical?

25–35 cycles