Question

18. When attempting a targeted gene knockout using mouse embryonic stem cells, there are three possible outcomes: targeted knockout, ectopic insertion, and no insertion. What procedures can be used to select for cells that only have the targeted gene knockout? (9 pts)

19. Describe why Jacob and Monod used IPTG as a synthetic inducer during their experiments investigating the genetic control of the lac operon. (6 pts)

20. Describe the function of the CAP-cAMP system in bacteria. Why does it regulate several operons related to metabolism? (7 pts)

21. Many DNA-binding proteins that affect gene expression are allosteric. What is allostery and why is it such a common mechanism of gene regulation? (7 pts)

22. A number of mutations affect the expression of the lactose operon in E. coli. Complete the table given below. Use + to indicate that a functional enzyme is synthesized and - to indicate that a functional enzyme is not synthesized. (12 pts)

	Inducer (IPTG) absent		Inducer (IPTG) present
	b-Galactosidase	Permease	b-Galactosidase	Permease
a) I+ O+ Z+ Y+
b) IS O+ Z+ Y+
c) I– O+ Z+ Y+
d) I+ OC Z+ Y+
e) IS OC Z+ Y–
f) I+ OC Z+ Y–/I– O+ Z– Y+

Answer 1

---Knockout organisms are also used as screening tools in the development of drugs, to target specific biological processes or deficiencies by using a specific knockout such as targeted knockout, or to understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae.

---The effect of lac operon in ther induction using lactose for the expression of periplasmic human interferon alpha2b was studied in shake flask culture.Escherichia coli strans Rosetta2 containing the deletion of entire lac operon with high level of lac repressor were used,R2 over expressed PrIFN-alpha2b at substantial levelin lactose induced media.In spite of the deletion of lac operon in RG2,the cells exposed to lactose produced PrIFN-alpha2b absent in less quantity.Under similar condition,the percentage of IFN-alpha2b translocated into periplasm for cells induced with lactose was 43-57 and 15-30 in R2 and RG2 respectively.Equivalent level of PrIFN-alpha2b expressed was obtsined in RG2 induced by IPTG and R2 induced by lactose.

--- Allostery is defined as the condition of a protein (enzyme) in which the structure and activity of the enzyme is remodeled by the binding of a metabolic molecule at a site other than the chemically active one.

Effector molecules bind at the protein's allosteric sites, leading to the changes of protein activity. Allostery of protein or DNA then play their respective biological roles through similar signaling pathways in evolution or deleterious physiological process to cause diseases.

----Please comments in the comment box if any doubt.Thanks...

	Inducer (IPTG) absent b-Galactosidase	Permease	Inducer(IPTG)present b-Galactosidase	Permease
a) I+ O+ Z+ Y+	0	0	+	+
b) IS O+ Z+ Y+	_	_	_	_
c) I– O+ Z+ Y+	+	+	+	+
d) I+ OC Z+ Y+	+	+	+	+
e) IS OC Z+ Y–	+	+	+	+
f) I+ OC Z+ Y–/I– O+ Z– Y+	_	+	_ +