CHAPTER 5 Minyeong Choi - Bacterial Transformation & Polymerase Chain Reaction

What is the clinical significance of bacterial transformation in biotechnology?

bacterial transformation explains horizontal gene transfer mechanisms responsible for rapid antibiotic resistance dissemination, enables recombinant DNA technology for pharmaceutical production, and provides insights into genetic engineering approaches for human gene therapy

When did Griffith discover transformation?

1928

How did the mouse die in station 1?

live s-strain injected into mouse

What did the s-strain contain?

it has polysaccharide capsule enabling immune evasion

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

it lacks capsule, appears rough and uncapsulated

What happened in station 3 of Griffith’s experiment?

heat-killed s-strain injected into mouse and mouse survives

What happened in station 4?

heat-killed s-strain + live R-strain injected into mouse

mouse dies from pneumonia caused by live s-strain

How did the bacteria demonstrate genetic variation?

R-strain acquired virulence from dead S-strain DNA

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

S chromosome/DNA segment released from heat-killed bacteria contains capsule synthesis gene

Recipient Bacterium with R chromosome/DNA segment incorporates foreign DNA through cell membrane

Competent 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 in the transformation process?

Dividing bacterium replicates hybrid chromosome containing S strain capsule gene

Daughter cells inherit transformed genotype with capsule synthesis capability

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

What is the clinical significance of bacterial conjugation?

conjugation facilitates rapid dissemination of antibiotic resistance plasmids in clinical settings, enables bacteria to acquire multiple resistance genes simultaneously, and represents a critical target for developing conjugation-blocking therapeutics to combat multidrug-resistant infections

What are the different bacterial mating types?

F+ bacteria

F+ chromosomes

F- bacteria

F- chromosomes

Fertility factor

What is the fertility factor?

episomal DNA conferring donor ability

What is the conjugation bridge?

a temporary, protein-lined passageway that forms between two bacterial cells during bacterial conjugation

How are genes transferred during conjugation?

via cell to cell contact

What is high frequency recombination?

a special type of bacterium, often E. coli, where 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?

Bacteriophage recognizes specific receptor sites on bacterial surface

Phage DNA contained within icosahedral capsid structure

Bacterial Wall provides attachment site for phage tail fibers

Host-phage specificity determined by receptor-ligand complementarity

What happens during phage DNA replication?

DNA segment comprises fragmented bacterial chromosomal DNA

Phage DNA directs synthesis of phage-encoded 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 occurs when bacterial DNA segments mistakenly packaged

Phage protein coat assembles around bacterial DNA instead of phage DNA

Transducing particles contain bacterial genes rather than complete viral genome

Cell lysis releases mixture of normal phages and transducing particles

Any bacterial hene 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

Aberrant exicision removes phage DNA with flaking bacterial genes

What is hybrid DNA formation?

the process where two complementary single strands of DNA (or DNA and RNA) bond together via hydrogen bonds to form a double-stranded hybrid molecule

How is DNA transferred to recipient?

Transformation (uptake of free DNA), Transduction (via viruses/bacteriophages), and Conjugation (direct cell-to-cell contact using a pilus)

What is the pre-integration state?

a viral-host protein complex containing viral DNA that's ready to insert into a host cell's genome, but hasn't yet

What is the post-integration state?

homologous recombinant integrates donor DNA into host chromosome

host DNA now contains recombinant sequences from donor daughter cells

phenotypic occurs as recipient expresses donor genes

What is the clinical significance of bacterial transduction?

transduction mediates virulence factor transfer in Staphylococcus aureus and Streptococcus pyogenes, enables rapid dissemination of antibiotic resistance genes, and represents a critical mechanism for bacterial pathogen evolution in clinical environments

How did genetic engineering revolutionize medicine?

by enabling bacteria to produce human proteins like insulin, growth hormone, and blood-clothing

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 for genetic engineering

What are restriction enzymes?

molecular scissors that cleave DNA at specific recognition sequences

creates sticky ends that allow precise DNA fragment insertion

What are DNA ligase?

molecular glue that seals DNA strands together

operates during DNA replication and repair by forming phosphodiesterase bonds between nucleotides at the restriction sites

Where do foreign DNA come from?

the gene of interest isolated from various sources including other bacteria, animal tissues (mouse), or human cells

cleaved into fragments by the same restriction enzyme to ensure compatible ends

What is a chimera?

the recombinant plasmid created when foreign DNA combines with the donor plasmid at the restriction point

this hybrid molecule now carries genetic instructions from a different organisms

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?

through binary fission

How can recombinant proteins be used?

recombinant proteins can be harvested from bacterial cultures for medical use

How did PCR revolutionize medicine?

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?

deoxynucleotides: the building blocks for new DNA synthesis

What is Taq DNA polymerase?

taq polymerase enzyme that synthesizes new DNA strands

What are the three steps of PCR?

initialization/denaturation

annealing

elongation

What temperature does denaturation occur at?

95 degrees celsius

What happens during the denaturation phase?

sample is heated and high temp breaks hydrogen bonds between complementary base pairs

double-stranded DNA separates into two single strands

the separated template strands are now available for primer binding

What temperature does annealing happen?

55 degrees celsius

What happens during annealing phase?

primers hydrogen bond to their complementary sequences on single-stranded template DNA

forward and reverse primers now base-paired to template strands

short DNA sequences that define the boundaries of amplification

only primers with complementary sequences bind

What temperature does elongation happen at?

72 degrees celsius

What occurs during elongation stage?

DNA polymerase extend from each primer, synthesizing new DNA strands

shows DNA polymerase adding nucleotides

complementary strands built by DNA polymerase

synthesis always proceeds →

How does PCR multiply? (mathematical formula)

2^n

How many cycles of PCR are typical?

25-40 cycles