Question

1. You are attempting to identify genes involved in osmotic stress response in Bacillus atrophaeus, a Gram positive endospore former. For your work, you set up a chemostat (continuous) culture. You remove two samples of culture, expose one to a high salt concentration, wait five minutes, and then extract all their mRNA. You then label the control with a green fluorescent die, the experimental with a red fluorescent dye, and then hybridize the mixture to a microarray. Following analysis, the microarray reader generates a list of ratios of fluorescence of the spots on the array calculated as red vs. green. The expression ratios of the first five genes (of many thousands!) are as follows:

Gene A: 1.002

Gene B: 34.271

Gene C: 0.260

Gene D: 27.930

Gene E: 0.997

a. At what stage of growth are your cells in the chemostat?

b. Imagine you repeated the above experiment every day for a week. How might your results compare if done using cells from a continuous culture (as above) vs. from a batch culture? Explain.

c. Based on the microarray data, which gene(s) might be involved in stress response to salt?

d. Which gene(s) might be involved in constitutive (background, needed all the time) functions?

e. Which gene(s) might be involved in a process not essential to survival during times of stress?

f. Confident you have identified a number of genes involved in protecting B. atrophaeus from salt stress response, you decide to begin by studying one further. How might you genetically engineer B. atrophaeus to demonstrate the involvement of this gene in stress response to salt?

Answer 1

a. At what stage of growth are your cells in the chemostat?

Answer: Active logarithmic phase

b. Imagine you repeated the above experiment every day for a week. How might your results compare if done using cells from a continuous culture (as above) vs. from a batch culture? Explain.

Answer: continues culture will give the same result as presented here. whereas, batch culture may not predicts true expression value for transcripts as cells aged and die will not hold the active mRNA for analysis.

c. Based on the microarray data, which gene(s) might be involved in stress response to salt?

Ans: Gene B, Gene D highly expressed in the stress. Gene C repressed in the stress.

d. Which gene(s) might be involved in constitutive (background, needed all the time) functions?

Ans: Gene A, Gene E as there level of expression is not disturbed much.

e. Which gene(s) might be involved in a process not essential to survival during times of stress?

Ans: Gene A, Gene E as there level of expression is not disturbed much.

f. Confident you have identified a number of genes involved in protecting B. atrophaeus from salt stress response, you decide to begin by studying one further. How might you genetically engineer B. atrophaeus to demonstrate the involvement of this gene in stress response to salt?

Ans: We can mutate the genes which expression is found to be high or low and can check the phenotype for the cells and predict the experimentation further.