LS23L Lab C Week 3: b-Galactosidase Assay

For wild type E. Coli present in a glucose-free environment, adding a high level of ___ causes the ___ to ___ the operator which in turn ___ the breakdown of lactose. Select one: a. lactose; lac repressor; fall off; promotes b. lactose; lac repressor; bind; promotes c. glucose; RNA polymerase; bind; promotes d. glucose; RNA polymerase; bind; halts e. glucose; lac repressor; fall off; promotes

A

What type of blank should be used for cell density assay? Select one: a. LB blank provided b. ONPG blank c. water blank d. Z buffer blank

A

Why did we need to measure the absorbance at two wavelengths during the beta-galactosidase lab? Select one: a. At 600 nm the absorbance is proportional to cell density while at 420 nm the absorbance is proportional to the amount of o-nitrophenol produced. The concentration of o-nitrophenol can be measured because it is a blue compound. b. 420 nm yields the best estimate of o-nitrophenol concentration and 600 nm yields the best estimate of galactose concentration. c. The optical density at 420 nm gives us an estimate of the concentration of o-nitrophenol, a yellow compound, and OD600 provides an estimate of cell density, which is inversely proportional to beta-galactosidase activity. d. At 600 nm the absorbance is proportional to cell density while at 420 nm the absorbance is proportional to the amount of galactose produced. e. At 600 nm the absorbance is proportional to cell density, which helps standardize our enzyme activity calculations, while at 420 nm the absorbance is proportional to the amount of o-nitrophenol produced.

A

According to the Lab Safety Sheet provided for this week, when working with E. coli cells you must wear: (check all that apply) Select one or more: a. Standard lab attire b. Safety goggles c. Lab coat d. Gloves e. Full-face air purifying respirator

A, B, C, D,

All except which of the following reagents should be kept on ice when not being used? a. Z buffer b. Na2Co3 solution c. E coli cell culture d. LB + glucose solution e. ONPG solution

B

In the beta gal assay, it is important to measure the density of e coli cells because: a. the transformation efficiency of e coli cells is low b. e coli densities vary among cultures with time, which will affect estimates of b-gal production rates c. we are measuring per cell cleavage of x gal in relation to culture conditions and time d. ONPG production by e coli is a function of cell density e. the lac operon is induced only in a small percentage of cells in a culture

B

Microcentrifuge tubes with chloroform in the bottom must be: a. Capped and placed in the autoclave bin in the hood b. Capped and placed in the labeled hazardous waste bin in the hood c. Uncapped and placed in the autoclave bin in the hood d. Uncapped and placed in the labeled hazardous waste bin in the hood e. Disposed of in the regular trash

B

What is the purpose of sodium carbonate (Na2Co3) in this lab? a. Seperates the bonding between enzyme subunits b. Drastically changes the pH to stop the enzymatic reaction c. Provides nutrients for bacteria growth d. Provides an optimum pH for enzymatic reaction e. Acts as a substrate that gives a yellow color when cleaved

B

Which of the following is a potential hazard you will face in the B gal assay lab? Circle all that apply. a. Corrosive chemicals b. Potentially carcinogenic chemicals c. Chemicals causing respiratory tract, eye, and skin irritation d. water reactive chemicals e. none of the above

B, C

According to the Lab Safety Sheet provided for this week, which of the following is a potential hazard you will face in the b-galactosidase assay lab? (Check all that apply) Select one or more: a. Radioactive materials b. Chemicals causing respiratory tract, eye and skin irritation c. Corrosive chemicals d. Water reactive chemicals e. Potentially carcinogenic chemicals

B, E

In the "biochemical assay of b-galactosidase activity", what is o-nitrophenyl-b-D-galactosidase (ONPG) used for? Select one: a. It provides essential nutrients for bacterial growth. b. It partially disrupts the cell membrane to allow cellular proteins to diffuse out of the cell. c. When ONPG is cleaved by b-galactosidase, we are able to assay b-galactosidase activity. d. ONPG cleaves the b-galactosidase that is made by the lac operon so we can see how much activity there is in the cell. e. ONPG cleaves galactose so we can measure how much lactose there is in the cell.

C

What is the purpose of Luria broth in this lab? Select one: a. drastically changes the pH to stop the enzymatic reaction b. partially disrupts cell membrane to allow substrate to enter the cell c. provides nutrient for bacteria growth d. provides an optimum pH for enzymatic reaction e. separates the bonding between enzyme subunits Feedback

C

What is the purpose of chloroform in this lab? Select one: a. provides nutrient for bacteria growth b. separates the bonding between enzyme subunits c. partially disrupts cell membrane to allow substrate to enter the cell d. drastically changes the pH to stop the enzymatic reaction e. provides an optimum pH for enzymatic reaction

C

What type of blank should be used for the B-gal assay? a. Z buffer blank b. ONPG blank c. "b-gal" blank made by your group d water blank

C

Which of the following is not a component of the lac operon in E. coli? Select one: a. Lac Z gene (for beta-galactosidase) b. Operator c. Lac I gene (for Lac repressor) d. Promoter e. Lac A gene (for Thiogalactoside transacetylase)

C

You are conducting the beta gal assay lab and your TA unknowingly gives you a mutatnt E. coli strain that cannot produce functional lac repressor proteins. You grow the E. colin in the presence of glucose, in the presence of lactose, and in the absence of both sugars. At 70 min, you assay for beta gal activity using ONPG as the cleavable substrate. What results should you expect to find? a. beta gal activity can't be assayed because beta gal can't be produced by the mutant E-coli strain b. beta gal activity should be higher for the e coli grown in the presence of glucose c. beta gal activity should be equal for all three conditions d. beta gal should be higher for the e coli grown in the absence of lactose and glucose e. beta gal should be higher for the e coli grown in the presence of lactose

C

What is the purpose of Z buffer in this lab? a. Seperates the bonding between enzyme subunits b. Drastically changes the pH to stop the enzymatic reaction c. Provides nutrients for bacteria growth d. Provides an optimum pH for enzymatic reaction e. Acts as a substrate that gives a yellow color when cleaved

D

Based on the beta gal lab, beta gal activity depends on a. cell density b. temperature c.time d.the sugar substrate present e. time and sugar substrate present

E

What is the natural substrate of B-galactosidease? a. IPTG b. ONPG c. Glucose d. Galactose e. Lactose

E

What is the purpose of ONPG in this lab? a. Seperates the bonding between enzyme subunits b. Drastically changes the pH to stop the enzymatic reaction c. Provides nutrients for bacteria growth d. Provides an optimum pH for enzymatic reaction e. Acts as a substrate that gives a yellow color when cleaved

E

Why is ONPG, in addition to lactose used as a substrate to determine b gal activity a. it is an enzyme that enhances the activity of b gal b. turns the products of lactose yellow making it easy to detect c. ONPG and lactose are both the natural substrates of b gal in bacteria d. cleavage of lactose molecules is time dependent, whereas the cleavage of ONPG is not e. the amount of products from ONPG is easily calculated by measuring its optical density, whereas the amount of products form lactose is more difficult to detect

E

According to the Lab Safety Sheet for the b-gal. assay lab, it is okay to use chloroform at your bench work station as long as you always use the dropper bottle and wear gloves. T or F

F

According to the Lab Safety Sheet, in the b-galactosidase assay lab, used gloves should be disposed of in the autoclave bag. Select one: True False

T or F