Transposon mutagenesis of salmonella Flashcards
Characteristic of Salmonella enterica?
- Pathogenic
- rod shaped
- gram negative
- found in intenstinal lumen
- causes diarrhea, vomiting, fever
- its a phototroph
how is salmonella enterica spread?
through fecal contamination of foods
What is a phototroph?
its capable of synthesizing a wide variety of its nutrients including amino acids, nucleic acids, and vitamins from inorganic nutrients.
What are transposons?
they are mobile genetic elements
“jumping genes”
they can hop from one place to another in the host genome
What transposon will we be using in this experiment?
Tn5.
characteristics of unmodified Tn5?
- its 5818 base pairs long and contains three antibiotic resistance genes.
one gene will encode for kanamycin resistance. - flanked by terminal inverted repeats (is50L and is50R)
What is exconjugant
cells that have undergone conjugation and the transfer of genetic material
what is conjugation
transfer of genetic material (plasmid) between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells
What is transposant?
mutant in which the transposon has hopped onto the salmonella chromosome)
what is transconjugants?
cells that have received the plasmid by conjugation and have had the transposon hop onto the chromosome.
significance of is50R?
includes 2 genes:
- one for transposase
- one for inhibition of transposase
Significance of terminal inverted repeats (is50L and is50R)?
they are the site of location where the transposase recognizes, binds and cuts out the DNA segment
What is mini Tn5? and its characteristics?
- smaller version of Tn5
- lacks transposase gene in is50R
- contains a plasmid pRL27
what is pRL27 and the significance of pRL27?
its a plasmid that carries the mini transposon and can be transferred from one E.coli donor strain to any wide variety of recipient strains by conjugation
what happens to the pRL27 once inside a recipient cell?
the transposon hops into the chromosomal DNA at a high frequency and grants kanamycin resistance
contents of pRL27 plasmid?
- origin of replication for a plasmid (oriR6k)
- OriT site
- Tnp site
What is oriR6K dependent on?
it requires the presence of the pi protein for replication of the plasmid.
how do you get the pi protein?
its encoded by the pir gene and is present on the chromosome of the E.coli host strain.
What is OriT?
its the site where DNA is nicked before conjugal transfer of the nicked DNA strand.
What is Tnp?
it a gene that encodes the transposase enzyme responsible for movement of the transposable element.
What is Tnp under control by?
its under the control of the tetA promoter. Which is known to be active in a wide variety of bacteria.
what is the aph gene?
its a gene that encodes the enzyme responsible for kanamycin resistance and
2. is the selective marker
What is OriR6k and its position?
the position is inside the transposon (inside mini Tn5)
it replicates the plasmid
what will happen to the single nicked strand of DNA?
it will enter salmonella as a linear piece,
Can salmonella reproduce the pRL27 plasmid?
it cannot because it cant produce pi protein
What is the donor strain in transposon mutagenesis?
E.coli
characteristics of E.coli donor strain
lacks the ability to synthesisze DAP.
it is resistant to kanamycin because it carries the pRL27 plasmid.
it also has the pir gene and expresses pi protein required for replication of the pRL27 plasmid
what is DAP
its an amino acid analog that is part of the bacterial cell wall.
What is the recipient strain?
salmonella
characteristics of the Salmonella recipient strain?
- sensitive to kanamycin
2. able to produce DAP and can grow in media lacking DAP
what media is used for selection?
a medium that contains kanamycin
it kills the recipient strain
what media is used for counter selection?
a medium that prevents growth of the donor strain.
We use a medium that lacks DAP to select against E.coli
once the donor strain and recipient strain have been allowed to mate, what happens next?
the cells are then plated on a medium thats both selective and counter selective
(LB+kan, no dap)
what will this medium thats both counter selective and selective do to the cells?
- will kill all o the donor e.coli cells which need DAP to survive
- will kill the recipient salmonella cells that have not picked up the plasmid
what cells survive from the medium that is both counter selective and selective?
- the cells that have not only received the plasmid but have moved the kan resistance gene from the plasmid to the chromosome.
True or false: the recipient strain is unable to replicate the plasmid and will therefore be unable to pass it on to any daughter cells
True
What is the only colony that will grow on this counter selective and selective medium?
salmonella with a transposon hopped onto the chromosome
