P-5

Question 1

what are the experiments in passage 5

Experiment 1

Answer 1

host cells were incubated in cell growth media containing vibrio parahaemolyticus for 2 hours. the cells were rinsed and incubated in new media containing antibiotic effective against a wide range of bacteria including VP. At hoully intervals samples of host cells were collected and rinsed, intracellular bacterial levels were measured

Question 2

what is the second experiment in passage 5

Answer 2

To confirm that VopC( the catalytic domain of the toxin) activates Rac, a mutant strain of VP lacking the VopC gene was created (VP-vopC). plasmids encoding wild type VopC or catalytically inactive VopC (VopCX)were transfected into VP-vopC to create strains VP-vpoC +VopC and VP-vopC + VopCX , respectively . Host cells were exposed to each strain and GTP-bound Rac was measured by western blot

Question 3

What was the results in experiment 1 and why

Answer 3

after 4 hours the bacterial colony forming units were low

This is due to the host cells were lysed by the VP and the ones that were not lysed has low bacterial count

Question 4

what could be the negative control for experiment 1

Answer 4

host cells only, with out VP and checking the bacterial colony forming units hourly

Question 5

what could be the positive control for experiment 1

Answer 5

VP only with out host cells and counting the colony forming units

Question 6

what is the dependent variable

Answer 6

number of colony forming units

Question 7

In experiment 2 they talk about measuring GTP -bound Rac: What is Rac ?

Answer 7

Rac is a subfamily of the Rho family of GTPases:

These proteins has GTPases activity; there are 4 of them Rac1,Rac2,Rac3 and RhoG

Question 8

how did they measure the GTP bound Rac ?

Answer 8

western blot will seperate GTP bound Rac form non-GTPbound Rac and then ELISA can be used to detect GTP-bound Rac