Chapter 5: Bacterial Transformation + PCR

What is the clinical significance of bacterial transformation in biotechnology?

• Explains antibiotic resistance spread

• Enables genetic engineering for insulin production

• Underlies gene therapy vectors used in treating inherited diseases

When did Griffith discover transformation?

• 1928

How did the mouse die in station 1?

• Live S Strain injected into mouse

• Died from pneumonia

What did the S strain contain?

• Polysaccharide capsules

• Enables immune evasion

How was the R strain different from the S strain?

• Lacks capsule

• Appears rough + uncapsulated

What happened in station 3 of Griffith’s experiment?

• Heat-killed S strain injected into mouse

• Thermal denaturation destroys bacterial proteins

• Mouse survives, bacteria cannot replicate

What happened in station 4?

• Heat-killed S strain + live R strain injected

• Mouse dies from pneumonia

• Living, encapsulated S strain found in blood

• Demonstrates transformation

How did the bacteria demonstrate genetic variation?

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

• S chromosome released from heat-killed bacteria

  • Contains capsule synthesis gene

• Recipient bacteria with R chromosome incorporates foreign DNA through cell membrane

• Bacteria express surface receptors for exogenous DNA uptake

What happens during DNA integration?

• Nucleases degrade one DNA strand while complementary strand integrates via homologous recombination

• Integrated S DNA replaces homologous region of R chromosome

What happens during bacterial division?

• Dividing bacteria replicates the hybrid chromosome w/ S strain capsule gene

• Daughter cells inherit transformed genotype w/ capsule synthesis capability

• Capsule production in offspring creates disease producing S strain from harmless R strain

What is the clinical significance of bacterial conjugation?

• Drives antibiotic resistance in hospitals

• Enables horizontal gene transfer (accelerates bacterial evolution)

• Serves as a model for strategies targeting genetic exchange

What are the different bacterial mating types?

• F+ Bacteria

• F- Bacteria

What is the fertility factor?

• Episomal DNA conferring donor ability

What is the conjugation bridge?

• Sex pilus extends from F+ cell to establish physical contact

How are genes transferred during conjugation?

• Strand migrates through pilus

• Recipient F- cell synthesizes complementary strand to complete plasmid

What is high frequency recombination?

What is bacterial transduction?

• Bacteriophages transfer genetic material to bacteria

• Spreads virulence genes

• Serves as molecular tool for bacterial genetics and gene therapy vector development

What happens during bacteriophage attachment?

• Bacteriophage recognizes receptors sites on bacterial surface

• Bacterial wall provides attachment site for phage tail fibers

What happens during phage DNA replication?

• DNA segment comprises fragmented bacterial chromosomal DNA

• Phage DNA directs synthesis of phage-ended enzymes

• Replicating phage DNA uses bacterial machinery for viral genome amplification

• Multiple copies of phage DNA and protein coat components accumulate

What happens during packaging and phage release?

• Aberrant packaging is when bacterial DNA segments are mistakenly packaged

• Any bacterial gene can be transferred via random packaging

What are the steps of generalized transduction?

• Bacteriophage attachment

• DNA injection and fragmentation

• Phage DNA replication

• Packaging error and phage release

What happens during prophage integration and excision?

• Donor bacteria harbors integrated prophage DNA within chromosome

• Donor DNA includes both phage and adjacent bacterial genes

• Temperate phage integrates at specific attachment sites in bacterial chromosome

What is hybrid DNA formation?

• The formation of DNA w/ partial phage genome fused w/ specific bacterial genes

How is DNA transferred to recipient?

• Through phage infection

• Recipient bacteria undergoes homologous recombination w/ incoming DNA

• Hybrid DNA is integrated via site-specific recombination

What is the pre-integration state?

• Host bacteria contains recipient chromosome prior to integration

• Host DNA represents native bacterial genome sequences

• Transduced bacterial DNA segment enters via phage protein coat

W

What is the post-integration state?

• Homologous recombination integrates donor DNA into host chromosome

What is the clinical significance of bacterial transduction?

• Mediates virulence factor transfer in S. aureus and Strep. pyogenes

• Enables rapid dissemination of antibiotic resistance genes

• Represents mechanism for bacterial evolution

How did genetic engineering revolutionize medicine?

• We can use bacteria to produce human proteins

What is the donor plasmid?

• Small, circular DNA molecule

• Isolated from E. coli

What are restriction enzymes?

• Cutting enzymes

What are DNA ligases?

• Form phosphodiester bonds

Where do foreign DNA come from?

• Various sources such as

  • Bacteria

  • Animal tissues

  • Human cells

What is a chimera?

• Recombinant plasmid created when foreign DNA combines w/ donor plasmid at restriction point

What does CaCl₂ solution do to cells?

• Opens cell walls and membranes

• Permits chimera entry into bacterial cytoplasm

• Allows cooling and membrane resealing

How does the host bacteria reproduce quickly?

• Binary fission

How can recombinant proteins be used?

• A variety of ways that impact the medical field

How did PCR revolutionize medicine?

• Rapid covid testing

• Cancer mutation screening

• Forensic DNA analysis

• Paternity testing

• Detection of infectious diseases

What are primers?

• Easy

What are dNTPS?

• Easy

What is Taq DNA Polymerase?

• Easy

What are the 3 steps of PCR?

• Initialization/Denaturation

• Annealing

• Elongation

What temp does denaturation occur at?

• 95C

What happens during the denaturation phase?

• DNA is broken apart

What temp does annealing happen?

• 55C

What happens during annealing?

• Primers bind to template

What temp does elongation happen at?

• 72C

What occurs during elongation stage?

• Taq Polymerase at work

How does PCR multiply?

• 2ⁿ

How many cycles of PCR are typical?

• Cycle 30