Transposon mutagenesis of salmonella

Question 1

Characteristic of Salmonella enterica?

Answer 1

1. Pathogenic
2. rod shaped
3. gram negative
4. found in intenstinal lumen
5. causes diarrhea, vomiting, fever
6. its a phototroph

Question 2

how is salmonella enterica spread?

Answer 2

through fecal contamination of foods

Question 3

What is a phototroph?

Answer 3

its capable of synthesizing a wide variety of its nutrients including amino acids, nucleic acids, and vitamins from inorganic nutrients.

Question 4

What are transposons?

Answer 4

they are mobile genetic elements
“jumping genes”
they can hop from one place to another in the host genome

Question 5

What transposon will we be using in this experiment?

Answer 5

Tn5.

Question 6

characteristics of unmodified Tn5?

Answer 6

1. its 5818 base pairs long and contains three antibiotic resistance genes.
   one gene will encode for kanamycin resistance.
2. flanked by terminal inverted repeats (is50L and is50R)

Question 7

What is exconjugant

Answer 7

cells that have undergone conjugation and the transfer of genetic material

Question 8

what is conjugation

Answer 8

transfer of genetic material (plasmid) between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells

Question 9

What is transposant?

Answer 9

mutant in which the transposon has hopped onto the salmonella chromosome)

Question 10

what is transconjugants?

Answer 10

cells that have received the plasmid by conjugation and have had the transposon hop onto the chromosome.

Question 11

significance of is50R?

Answer 11

includes 2 genes:

1. one for transposase
2. one for inhibition of transposase

Question 12

Significance of terminal inverted repeats (is50L and is50R)?

Answer 12

they are the site of location where the transposase recognizes, binds and cuts out the DNA segment

Question 13

What is mini Tn5? and its characteristics?

Answer 13

1. smaller version of Tn5
2. lacks transposase gene in is50R
3. contains a plasmid pRL27

Question 14

what is pRL27 and the significance of pRL27?

Answer 14

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

Question 15

what happens to the pRL27 once inside a recipient cell?

Answer 15

the transposon hops into the chromosomal DNA at a high frequency and grants kanamycin resistance

Question 16

contents of pRL27 plasmid?

Answer 16

1. origin of replication for a plasmid (oriR6k)
2. OriT site
3. Tnp site

Question 17

What is oriR6K dependent on?

Answer 17

it requires the presence of the pi protein for replication of the plasmid.

Question 18

how do you get the pi protein?

Answer 18

its encoded by the pir gene and is present on the chromosome of the E.coli host strain.

Question 19

What is OriT?

Answer 19

its the site where DNA is nicked before conjugal transfer of the nicked DNA strand.

Question 20

What is Tnp?

Answer 20

it a gene that encodes the transposase enzyme responsible for movement of the transposable element.

Question 21

What is Tnp under control by?

Answer 21

its under the control of the tetA promoter. Which is known to be active in a wide variety of bacteria.

Question 22

what is the aph gene?

Answer 22

its a gene that encodes the enzyme responsible for kanamycin resistance and
2. is the selective marker

Question 23

What is OriR6k and its position?

Answer 23

the position is inside the transposon (inside mini Tn5)

it replicates the plasmid

Question 24

what will happen to the single nicked strand of DNA?

Answer 24

it will enter salmonella as a linear piece,

Question 25

Can salmonella reproduce the pRL27 plasmid?

Answer 25

it cannot because it cant produce pi protein

Question 26

What is the donor strain in transposon mutagenesis?

Answer 26

E.coli

Question 27

characteristics of E.coli donor strain

Answer 27

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

Question 28

what is DAP

Answer 28

its an amino acid analog that is part of the bacterial cell wall.

Question 29

What is the recipient strain?

Answer 29

salmonella

Question 30

characteristics of the Salmonella recipient strain?

Answer 30

1. sensitive to kanamycin

2. able to produce DAP and can grow in media lacking DAP

Question 31

what media is used for selection?

Answer 31

a medium that contains kanamycin

it kills the recipient strain

Question 32

what media is used for counter selection?

Answer 32

a medium that prevents growth of the donor strain.

We use a medium that lacks DAP to select against E.coli

Question 33

once the donor strain and recipient strain have been allowed to mate, what happens next?

Answer 33

the cells are then plated on a medium thats both selective and counter selective
(LB+kan, no dap)

Question 34

what will this medium thats both counter selective and selective do to the cells?

Answer 34

1. will kill all o the donor e.coli cells which need DAP to survive
2. will kill the recipient salmonella cells that have not picked up the plasmid

Question 35

what cells survive from the medium that is both counter selective and selective?

Answer 35

1. the cells that have not only received the plasmid but have moved the kan resistance gene from the plasmid to the chromosome.

Question 36

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

Answer 36

True

Question 37

What is the only colony that will grow on this counter selective and selective medium?

Answer 37

salmonella with a transposon hopped onto the chromosome