chapter 8.1 case study, mechanism responsible for glucose-lactose diauxie in Escherichia coli: challenge to the cAMP model

aim

study the role of cAMP in repressing lactose metabolism in the presence of glucose in E.coli.

hypothesis

cAMP is involved in promoting lactose metabolism when there is little to no glucose because it helps activate the CAP protein

background

Beta-galactosidase:

• enzyme found inside the lacZ gene in the lac operon

• Beta gal levels indicate activation and expression of the whole operon. This is because the lacZ gene in the operon means that if that gene is expressed, the whole operon is expressed, so the trends that you see in beta gal will be the same for everything in the operon

Glycerol:

• bad alternative C source for E.coli. glycerol can be used to grow but it is not very good (recall// glucose is primary)

What was the purpose of the glycerol treatment?

• It is the control: without lactose and glucose

• To prove that when there is no glucose or lactose, bacteria can still grow

Considering what you know about the lac operon model today, what is unexpected in terms of the beta-galactosidase activity? Which treatments don’t meet the expected results?

Considering what you know about the lac operon model today, what is unexpected in terms of the cAMP levels? Which treatments don’t meet the expected results?

• In only glucose, there should be low levels of cAMP and no expression of the lac operon. This is shown in 0.4% glucose

• In only lactose, there should be high levels of cAMP (no glucose) and there is expression of the lac operon because lactose binds to the LacI repressor protein, so RNA pol is able to bind to the promoter. In 0.4% lactose beta gal activity is true, BUT cAMP COUNTS ARE UNEXPECTED. The levels should be high because there is no glucose present

• In both glucose and lactose there should be low levels of cAMP and no expression of the lac operon because there is the presence of glucose and since glucose is the preferred C source the conditions will be for glucose initially.

beta gal: expected for everything

cAMP: not expected for 0.4% lactose

According to the data, what condition(s) trigger expression of the lac operon?

• High levels/concentration of lactose

5. According to the data, what condition(s) trigger increase in cAMP levels?

Low levels/ concentration of glucose

conclusion

cAMP is always present and the CAP is always bound to the CAP site, REGARDLESS of glucose levels. This always enhances the lac operon

• Now know this is not true