genet quiz 3
Why do we add homology arms (H1 and H2) to the PCR product?
They ensure the PCR fragment recombines precisely at the intended genomic location via homologous recombination.
Where does the antibiotic resistance cassette come from?
From the template plasmid used in PCR (e.g., pKD3, pKD4). It is NOT from the bacterial genome.
Why can’t E. coli recombine linear DNA on its own?
Linear DNA signals phage infection → E. coli uses RecBCD to rapidly destroy it.
What does Gamma do?
Blocks RecBCD so the linear PCR DNA isn’t degraded.
Why is Exo needed?
Exo chews back 5′ ends → creates 3′ ssDNA overhangs required for Beta to anneal to the chromosome.
What does Beta do?
Binds the 3′ ssDNA overhangs → promotes strand invasion and annealing during recombination.
Why must arabinose be added BEFORE electroporation?
It induces pKD20 to express λ Red proteins so that the PCR fragment can recombine immediately after entering the cell. Without induction → no recombination → fragment degraded
What happens to linear PCR DNA that enters the cell but does NOT recombine?
After induction stops:
Lambda Red proteins decay
RecBCD becomes active again
Leftover linear DNA is destroyed
It cannot replicate → cannot form colonies
Why is the AbR cassette included even though we remove it later?
It acts as a temporary selectable marker so we can identify cells where recombination succeeded.
Why remove the AbR cassette later?
To:
Allow reuse of the same antibiotic marker
Prevent unwanted polar effects
Keep the genome clean
Enable future plasmid transformations without confusion
Why do FRT sites matter?
They are recognized by FLP recombinase which excises the AbR cassette during the flip-out step.
What’s left behind after flip-out?
A single FRT scar (~80 bp).
How do we verify the deletion?
Two ways:
Phenotypic: Patch onto LB vs LB+Ab → Ab-sensitive = AbR cassette gone
Genotypic: PCR → flip-out strain gives smaller band than WT
Why does the WT band appear higher on the gel than the Δgene band?
WT still contains the full gene → longer PCR product → slower migration → higher on gel.
Δgene only has a small FRT scar → smaller fragment → lower on gel.
What is pKD20 and does it insert into the chromosome?
pKD20 is a helper plasmid expressing Exo/Beta/Gamma.
It never integrates — it stays separate and is later cured.
Why use electroporation for the PCR product?
Linear DNA cannot enter efficiently via heat shock.
Electroporation creates pores allowing it to enter.
Why do we not see false positives from leftover PCR fragments?
Because:
After induction ends, Gamma disappears
RecBCD degrades all linear DNA
Only recombined fragments (inside the chromosome) survive
What happens if homology arms are too short?
Recombination efficiency drops dramatically or fails completely.
What happens if arabinose is added too late?
PCR fragment is degraded before recombination can occur → no colonies.
Why does this system only work in strains carrying pKD20?
Because λ Red proteins (Exo, Beta, Gamma) are essential for recombination and E. coli does not express them naturally.
What is the CTX element?
A DNA segment in toxigenic V. cholerae that includes ctxAB (cholera toxin genes) plus recombination sites (RS/att) used for integration/excision.
How did they track movement of the CTX element?
By replacing ctxAB with a KanR cassette, so any strain that receives CTX-Km becomes kanamycin resistant (Kmᴿ).
What is a supernatant?
The liquid above the pellet after centrifugation/filtration that contains secreted molecules & phages but no cells.
Why is conjugation ruled out?
Transfer still occurs when donor cells are removed and only the cell-free supernatant is used, so there is no cell-to-cell contact. Conjugation needs direct contact.
Why is transformation ruled out?
DNase I treatment (which destroys naked DNA) does not block transfer. So the transferred material is not free DNA(not transformation).
Why does heat or chloroform treatment point to phage?
Heat/chloroform destroy phage particles, and under these treatments transfer stops. This indicates transfer is via a phage (CTXφ).
What is the replicative form (RF) of CTXφ?
The double-stranded DNA form of the phage genome inside the host, called pCTX-Km when it carries the KanR replacement.
Why did they transform pCTX-Km into Peru-15?
To produce pure CTXφ particles in a host with no prophages and no TCP, so phage can be made but not re-infect the same cells. This gives a clean phage prep for EM and further experiments.
What did EM show about CTXφ?
It’s a filamentous phage, like M13 – long, thin, flexible filaments.
What does mitomycin C do in this context?
It causes DNA damage, triggering prophage induction. CTXφ excises from the chromosome, forms phage particles, and can then infect new cells.
How do they know mitomycin C induces CTXφ?
Supernatant from SM44 grown with mitomycin C contains more KmR-transducing particles which can convert O395 to Kmᴿ.
Why did they introduce 4 bp mutations in zot and orfU?
Tiny 4 bp insertion/deletion causes a frameshift → nonfunctional protein, but doesn’t delete the gene or its regulation completely. Good way to test if the proteins are needed.
What did the zot/orfU mutants show?
Mutated pCTX-Km plasmids no longer produced KmR phage particles, so zot and orfU are required for phage particle morphogenesis.
What is the receptor for CTXφ on V. cholerae?
TCP (toxin co-regulated pilus). Only TCP+ strains become Kmᴿ when exposed to CTX-Kmφ. TCP mutants are resistant.
What happens if a strain doesn’t express TCP?
CTXφ cannot bind or infect it → no transductants (no Kmᴿ colonies).
Why was O395 efficiently transduced both in vitro and in vivo?
O395 (classical) expresses TCP well both in vitro and in vivo, so CTXφ can infect it in lab media and in the mouse intestine.
Why did Bah-2 only get CTXφ in vivo?
Bah-2 (El Tor) expresses TCP mainly inside the host (in vivo), not well in vitro. So CTXφ could infect it in mice, but not efficiently in regular lab broth.
What is lysogenic conversion in this context?
When CTXφ integrates into a previously non-toxigenic V. cholerae strain, giving it cholera toxin genes and turning it into a pathogen.
What are “moron” genes?
Extra genes in a prophage genome (like ctxAB) that are not needed for the phage’s own life cycle, but provide advantages to the host, like virulence factors. (“More-on” the genome than necessary.)
Why is CTXφ important for public health?
Because it can move toxin genes between strains, leading to new toxigenic V. cholerae variants and possibly new pandemics.