Don't bother to revise! Flashcards
what is a suicide plasmid?
a plasmid which is replication incompetent
suicide plasmids contain an ORI that is NOT compatible with host cell machinery
a suicide plasmid will be lost from the cell as it divides unless
it integrates into the chromosome
how to make a suicide plasmid (4)
- identify target gene
- create a PCR primer that matches a section of the gene
- amplify it
- remove the gene and place it into a suicide plasmid + an antibiotic resistance gene
suicide plasmids are introduced into a cell through
transformation
how are bacterial genes investigated?
deleting genes and observing the phenotype
what are the two ways suicide plasmids can be transferred?
- Conjugation
Donor and recipient strains are mixed together - Electroporation
A brief electrical pulse is applied to the mixture, which increases the permeability
When a suicide plasmid is introduced to the recipient cell the cell is called a
Transconjugant (transient, cannot be maintained in the cell)
how does a transconjugant integrate into the host genome?
homologous recombination
integrated plasmid disrupts the gene
the fact that a suicide plasmid inactivates the gene it is inserted into is called
insertional inactivation
insertional inactivation can cause
polar effects
what are polar (downstream) effects?
Insertional inactivation of one gene within an operon may affect function of downstream genes within the same operon – “polar effects”
means you don’t know whether the phenotype is from the gene you disrupted or from polar effects
unnmarked deletion mutants is preferred
what is an unmarked deletion mutant?
a mutant where the gene inserted does not disable the gene
clone fragments that flank the target gene upstream and downstream
still clone into suicide plasmids, still contain a AB resistance marker AND a counterselectable marker
how to identify a deletion mutant
select for the AB resistance cells
then select for counterselectable marker sacB
sac B encodes a levansucrase enzyme that converts sucrose to a toxic product that is lethal
when plated onto sucrose, cells that have not undergone homologous recombination and thrown off sacB will die
upstream recombination results in WT (target gene still present)
downstream recombination results in loss of target gene, sacB and AB resistance genes
pros of unmarked deletions
no polar effects
no AB resistance
multiple genes can be deleted together
cons of unmarked deletions
time consuming
difficult to counter select
