Lab 3: b-galactosidase Assay

According to the Lab Safety Sheet provided for this week, when working with E. coli cells you must wear:

-Lab Coat -Standard lab attire -Gloves -Safety goggles

Which of the following procedures should be done in the hood?

Add chloroform into tubes for enzymatic assay

What type of blank should be used for the enzyme assay?

B-gal blank

What does the Z gene on the Lac operon code for?

Beta Galactosidase

Which of the following is not a component of the lac operon in E. coli?

I gene (for Lac repressor)

b-galactosidase is synthesized by E. coli grown in which of the following media?

LB + lactose

Why is ONPG, in addition to lactose, used as a substrate to determine B-galactosidase activity?

The amounts of products from ONPG is easily calculated by measuring its optical density, whereas the amount of products from lactose is more difficult to detect

When both glucose and lactose are present in abundance in E. Coli,

Transcription of the lac operon is turned off.

For the biochemical assay of B-Galactosidase Activity, why is the absorption at 600nm greater at 70 minutes compared to 20 minutes?

there are more cells.

What type of blank should be used for the b-galactosidase enzyme activity assay?

"b-gal blank" made by your group

Which of the following statements is true about ONPG used in the biochemical assay lab?

-ONPG has a similar structure to lactose. -B-gal cleaves ONPG to produce o-nitrophenol, which is a yellow compound that can be used as an indicator of b-galactosidase activity.

What is the purpose of chloroform in this lab?

-To allow cellular proteins to diffuse out of the cell. -partially disrupts the cell membrane allowing cellular proteins to diffuse out of the cell

You and your lab partners decide to redo the b-galactosidase lab, only this time, you incubate your E. Coli cultures for 15, 30, and 45 minute time points. After adding the PopCulture and the Z-buffer, you add ONPG and incubate for 20 minutes. All other steps remain the same. When calculating the Units of Enzyme Activity at the end of the lab, what value would you use for the "time" variable?

20 Minutes

Which of the following is NOT TRUE regarding the lac operon? a. Gene O is the lac repressor binding site. b. As glucose concentrations increase, lactose concentrations decrease. c. Active beta-galactosidase breaks down lactose into glucose and glycine. d. Lactose binds to the repressor protein, causing an allosteric change. e. Gene I encodes for the lac repressor.

Active beta-galactosidase breaks down lactose into glucose and glycine.

Chemicals/materials you will use in the b-galactosidase assay lab include:

All of these

In lab, B-galactosidase enzyme activity was measured in "units of activity". In order to determine the units of activity, one must be able to measure a. the volume of the sample assayed. (volume) b. E. coli density. (OD600) c. how long the reaction between B-galactosidase and its substrate was allowed to proceed. (time) d. the concentration of o-nitrophenol. (OD420)

All of these.

When lactose is the only carbon source available, what effect does it have on the Lac repressor protein?

Allolactose binds to the repressor protein, causing it to change shape and fall off the operon on the lac operon

In the B-galactosidase activity lab, you accidentally forget to add chloroform to your bacteria samples, both at 20 and 50 minutes of cell incubation. What ramifications will most likely occur in your spectrophotometer readings?

An increase in the 600 nm absorbance and no change in the 420 nm absorbance.

If a student completes the lab correctly, but forgets to add chloroform to his cells in the LB + lactose solution, what results should he expect at the end of the lab?

B-gal will be transcribed and the solution will be colorless (the cell will remain closed and the proteins will not be released from solution)

Why would an E. Coli culture produce more Beta Galactosidase, in an environment where there is lactose as opposed to an environment where there is lactose and glucose? What specific gene in an E. coli's genome is transcribed in both environments?

Because cAMP binds to the CAP protein which can then bind to the CBS (cap binding site) to enhance the RNA Polymerase catalyzed transcription of lac Z gene into mRNA which is translated into beta-Galactosidase. The I gene is transcribed in both cases.

If the lac repressor of E. coli is mutated so that it never binds to the operator. Which of the following would be true? a. Glucose digesting enzymes are never produced b. Beta galactosidase is always produced c. The result depends on the concentration of glucose d. The result depends on the concentration of lactose e. Beta galactosidase is never produced

Beta galactosidase is always produced

What is the normal function of the protein product encoded by the I gene?

Binds to the operator of the lac operon (the repressor is encoded by the lac I gene)

In the B-galactosidase lab, what is NOT in the equation for calculating enzyme activity?

Carbon source.

The organism in which this enzyme will be analyzed in this lab is ________

E. coli

Which of the following should NOT be added to the B-galactosidase blank? a. Z buffer. b. Na2CO3. c. ONPG. d. Chloroform. e. E. coli cells.

E. coli cells.

In the B-galactosidase assay, it is important to measure the density of E. coli cells because

E. coli densities vary among cultures and with time, which will affect estimates of b-galactosidase production rates.

According to the Lab Safety Sheet for the b-galactosidase 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.

False

In the b-galactosidase assay lab, used microcentrifuge tubes and pipette tips contaminated with E. coli should be disposed of in the regular trash.

False

In petri dish A, E. coli cells are grown in the presence of glucose as the only carbon source. In petri dish B, E. coli cells are grown in the presence of lactose as the only carbon source. Which of the petri dishes would have production of Beta-galactosidase? Which of the dishes would have an inactivated Lac repressor?

In petri dish B, Beta-galactosidase is synthesized and the Lac repressor is inactivated.

What type of blank should be used for cell density assay?

LB blank provided

Units of enzyme activity

OD420/(time x volume x OD600)

You use ONPG to assay b-galactosidase activity in two E. coli cultures. OD420 values indicate similar concentrations of b-galactosidase in both cultures. You do not measure OD600. You can conclude that

ONPG was cleaved at approximately the same rate in both cultures.

To measure the amount of enzyme activity in a cell, we use a procedure called a biochemical assay. To test the activity of b-galactosidase in E. coli cells, why did we need to add ONPG to the three sample tubes?

ONPG, a derivative of lactose, can be cleaved by b-galactosidase producing a yellow compound making it possible to assay enzyme activity

In the absence of lactose, the lac repressor can bind to __________ and can prevent the transcription of the __________.

Operator; Lac Z, Y, & A

What is the purpose of Z buffer in this lab?

Provides an optimum pH for enzymatic reaction

All of the following are true regarding the regulation of transcription of the lac operon except: a. Allolactose causes the lac repressor to change comformation so it gets off of the operator. b. RNA polymerase is stopped by the lac repressor when no lacotse is present. c. RNA Polymerase initially attaches to the operator. d. The transcribed I gene leads to the creation of mRNA that encodes the Lac repressor protein. e. The transcribed Z gene leads to the creation of mRNA that encodes beta-galactosidase.

RNA Polymerase initially attaches to the operator.

In the absence of lactose, what would occur if there was a mutation in the operator of the lac operon that did not allow the lac repressor to bind?

RNA polymerase would bind to the promoter, and would also transcribe B-galactosidase.

The bacteria grown in Luria broth with only lactose were expected to:

Show marked B-galactosidase activity.

A particular E. coli cell contains a lac operon that continuously breaks down lactose into glucose and galactose. What could be a possible explanation for this occurrence?

The I gene in the lac operon is mutated and does not encode for the lac repressor.

In the absence of lactose, which of the following could occur?

The I gene produces a repressor which inhibits the lac operon from transcribing beta-galactosidase.

A class is studying b-galactosidase activity in a bacterial culture. After a student adds ONPG to their sample, the student adds two drops of chloroform, mixes the samples, and allows the samples to sit before transferring the supernatant to a cuvette for a enzymatic assay reading using a spectrophotometry. To the student's surprise, their enzymatic assay reading was significantly lower than the rest of the class's. What is the best explanation for this?

The student added ONPG to the bacterial cells before lysing the cells. This does not allow the b-gal to come into contact with ONPG, which may have also been damaged by its direct exposure to chloroform.

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

True

Which of the following tells us the cell density in a solution?

absorbance reading at 600nm, and convert it to concentration.

In the b-galactosidase assay, cleavage of ONPG by b-galactosidase was terminated by

adding sodium carbonate (Na2CO3).

What is a conclusion that can be gained from the lab on beta-galactosidase?

beta-galactosidase synthesis does not require glucose and is time dependent

Microcentrifuge tubes with chloroform on the bottom must be

capped and placed in the labeled hazardous waste bin in the hood

To determine if the presence of glucose in the growth medium induces b-galactosidase synthesis, one can

compare the b-galactosidase activity in the presence of glucose to that in the absence of a sugar substrate.

In the beta-galactosidase assay, units of enzyme activity

described the amount of beta-galactosidase produced per E. coli cell per minute.

What is the purpose of Na2CO3 in this lab?

drastically changes the pH to stop the enzymatic reaction

What is the natural substrate of beta-galactosidase?

lactose

In the b-galactosidase assay lab, the readings of each sample at 420nm using the spectrophotometer measures the amount of

o-nitrophenol in sample.

What is the purpose of Luria broth in this lab?

provides nutrient for bacteria growth

The function of B-galactosidase is to break down lactose in E. coli. Which one of the followings is NOT a critical factor in determining the enzyme activity of B-galactosidase in a biochemical assay? a. Optical Density at 420 nm for the end product of breaking down ONPG. b. Time dependent for cleaving to complete. c. Amount of Luria Broth provided in the test tube. d. Optical Density at 600 nm for the cell density of E. coli bacteria. e. Volume of the sample containing E. coli

Amount of Luria Broth provided in the test tube.

We know that Beta-galactosidase activity is regulated by an operon that relies on the presence of lactose to remove the suppressor. However, in the presence of glucose and lactose, what would the expected activity be?

Beta- galactosidase activity will be hindered since the cell does not want to waste energy breaking down lactose, when glucose is readily available

ONPG, which is a derivative of lactose, is cleaved by beta-galactosidase into which of the following two compounds.

Galactose and o-nitrophenol

The lac operon regulates bacterial transcription by: I. Using lactose to block RNA Polymerase, and inhibit transcription II. Using glucose to block the Repressor which allows transcription III. Using allolactose to bind to the repressor causing an allosteric change and allow transcription.

III only

In the absence of lactose, what gene(s) on the lac operon is/are transcribed?

Lac I gene

Which of the following statements regarding the lac operon is false? a. When glucose is available, the lac operon undergoes negative control. b. Transcription of the lac genes produces a monocistronic message due to the fact that it occurs from a single promoter. c. An operator constitutive (Oc) mutation is defective in binding repressor and yields lac products in the absence of lactose. d. The repressor is also known as an allosteric protein. e. A basal level of the lac operon products is always present during repression.

Transcription of the lac genes produces a monocistronic message due to the fact that it occurs from a single promoter.

Why did you need to measure cell density at the 20-minute point as well as at the 70-minute time point?

We need to measure cell density at both time points in order to determine if the induction of B-galactosidase synthesis in E. coli is time-dependent.

You are assaying b-Galactosidase activity in a sample of E. Coli growing in LB with just lactose for 2 hours, but the spectrophotometer show that no significant amount of yellow o-nitrophenol was detected. Assume the spectrophotometer is also functioning properly, and the answer choices below depict the only error, which of the following errors is the best explanation?

You forgot to add chloroform.

If the I gene in the Lac Operon is mutated and can not get transcribed, the B-galactosidase level of a bacteria in glucose (and no lactose) is ______________.

high, because there is no Lac repressor blocking the RNA polymerase.

If one wants to measure B-galactosidase activity under different conditions, it is important to take cell density into account primarily because:

if cell density were not measured, one would not know whether more enzyme is being produced because of upregulation, or because of there just being more cells.

What is the purpose of PopCulture (choloform) in this lab?

partially disrupts cell membrane to allow substrate to enter the cell

When lactose is present which of the following occur together:

repressor is inactivated; transcription occurs

The ____________ will bind to the ____________ in the ____________ (presence/absence) of lactose

repressor, operator, absence

What is the purpose of ONPG in this lab?

substrate that gives a yellow color when cleaved (acts like lactose)

In the b-galactosidase assay, E. coli cell density was determined against

the Luria broth (LB) blank to account for the background absorbance of LB in the sample.

Time

the length of time that the reaction of B-gal and ONPG is permitted to proceed

While preparing samples for an experiment measuring b-galactosidase activity in E. coli cells, a student accidentally introduces both glucose and lactose into a culture medium. Assuming that b-galactosidase activity was monitored at a constant rate, what would a graph of enzymatic activity look like?

the level of b-galactosidase activity would be low as glucose is consumed, then rise as the E. coli began to consume lactose

Which of the following is false regarding the lac operon: a. In the absence of inducer (lactose), the operon is turned on. b. Repressor protein has two binding sites one for operator and one for the inducer (allolactose). c. The repressor protein has the control of the system and it can turn the operon off. d. Promoter is the region that binds RNA polymerase. e. The three genes Z, Y, and A encodes for three different enzymes.

In the absence of inducer (lactose), the operon is turned on.

In the presence of lactose, the Lac repressor is ____________, which allows the transcription of the lac operon to occur. Thus when lactose is the only source of sugar, the activity of the b-galactosidase is ____________ compared to the condition where glucose is the only source of sugar.

Inactivated, increased

In testing for b-galactosidase, why do we see a low level activity of the enzyme at the 20 minute time interval and a much higher activity of the enzyme at the 50 minute time interval when lactose is present?

It takes time for the bacteria to be induced into making the enzyme.

The _________________ binds to the ____________ and prevents the RNA polymerase from transcribing the ___________.

Lac repressor, operator, Lac Operon

Which of the following statements is considered incorrect? a. The enzyme beta-galactosidase catalyzes the hydrolysis of lactose into galactose and glucose. b. If all the enzymes that a cell will possibly need are produced all the time, then a cell will waste a lot of energy. c. Lactose is the only energy source that bacteria can utilize. d. When lactose is present, it binds to the repressor region of the lac operon, thereby causing a conformational change of the repressor and making it fall off from the operator region. e. The transcription of beta-galactosidase is regulated by the presence or absence of lactose in the environment.

Lactose is the only energy source that bacteria can utilize.

In the second lab, we investigated B-galactosidase activity of E. coli cells. During the B-Galactosidase assay, we added chloroform to the tubes. If one of the tubes contained E. Coli cells, B-Galactosidase, Lactose, Z-buffer; adding which of the following to the tube would give us the same result as adding chloroform?

Lactose permease

What does RNA polymerase transcribe from the Lac Y gene on the lac operon?

Lactose permease

The enzyme beta-galactosidase breaks down ______ into simpler sugars ________ and ________ .

Lactose, glucose, galactose..

The transcription of beta-galactosidase is dependent on the presence of which of the following compound?

Lactose.

An assay of which of the following solutions will theoretically have the highest amount of b-galactosidase enzyme activity? a. Luria Broth, glucose and lactose, at 20 mins of incubation b. Luria Broth & glucose, at 20mins of incubation c. Luria Broth and lactose, at 70 mins of incubation d. Luria Broth and lactose, at 20 mins of incubation e. Luria Broth, glucose and lactose, at 70 mins of incubation

Luria Broth and lactose, at 70 mins of incubation

All except which of the following reagents should be kept on ice when not being used?

Na2CO3 solution

If equal amounts of lactose and glucose are present, will there be b-galactosidase activity within 10 minutes of incubation?

No because glucose is the preferred sugar source.

In the Biochemical Assay of B-Galactosidase Activity lab, you will be using the spectrometer. Which of the following statements is false regarding the spectrometer in this lab? a. The absorption of a sample at 600nm is used to determine the cell density. b. A blank should have everything except the compound of interest which absorbs light. c. Your B-Gal blank should be used before measuring the optical density of o-nitrophenol. d. The absorption of a sample at 420nm is used to determine B-Galactosidase Activity. e. The Luria broth blank should be used after measuring the optical density of o-nitrophenol.

The Luria broth blank should be used after measuring the optical density of o-nitrophenol.

John wants to measure the activity of B-galactosidase using the same protocol followed by LS3 students. He performs the experiment correctly but instead of ONPG he mistakenly adds galactose to the cells after the chloroform step. How will his spectrophotometry results compare to those obtained with ONPG? a. The OD600 will be higher because ONPG stops cell growth. b. The OD600 will be lower because the cells need glucose to survive. c. The OD420 will be higher because galactose is a product of B-galactosidase activity. d. The OD420 be the same because B-galactosidase is not produced in the presence ofgalactose. e. The OD420 be lower because it measures the presence of products from the breakdown of ONPG.

The OD420 will be lower because it measures the presence of products from the breakdown of ONPG

Which of the following statements is false? a. The Lac operon's activity can be measured by the amount of beta-galactosidase present. b. The operator site of the Lac operon is a key factor in transcription of Lactose permease. c. The Lac operon is inactivated by the presence of lactose. d. The RNA polymerase transcribes Lac Z genes which code for lactose. e. The Lac repressor is encoded by the product of the I gene.

The RNA polymerase transcribes Lac Z genes which code for lactose.

The lac operon is devised to activate the transcription of the enzyme beta-galactosidase when needed. What conditions begin this process?

The lac repressor is deactivated through allosteric change in the presence of lactose allowing for transcription of the Z Gene to beta-galactosidase.

In the b-galactosidase lab, you used a spectrophotometer that had a fixed wavelength. What is the purpose of the spectrophotometer and why was the wavelength fixed at 420 nm?

The spectrophotometer measures the amount of light of specific wavelengths absorbed by a solution. Since the color of the solution was yellow, it will absorb a wavelength in the complementary region which is violet.

When lactose is present in E. Coli, which of the folllowing is false. a. galactose concentration in the bacteria would increase b. only the gene encoding beta-galactosidase would be transcribed c. glucose concentration in the bacteria would increase d. lactose binds to the lac repressor and allows transcription e. lac repressor changes conformation and falls of the operator

lactose binds to the lac repressor and allows transcription

A sample of E. coli would exhibit maximum B-galactosidase activity in the presence of which combination of nutrients?

luria broth and lactose

If lactose is the sole sugar source present in E.coli, then the repressor cannot bind to the _______ and lactose is broken down into _________ .

operator, glucose and galactose

In a biochemical assay, the transcription of b-galactosidase is monitored in bacterial cells. A substrate that functions like ONPG, however produces a purple color when cleaved, is added to the reaction mixture. Under a constant pH of 7, if b-galactosidase is transcribed in these cells,

the substrate will be cleaved as the mixture produces a purple color

In the b-galactosidase lab, one would expect that:

the tube containing LB + lactose cells turns yellow because beta- galactosidase cleaves ONPG.

Based on the beta-galactosidase lab, beta-galactosidase activity depends on

time and the sugar substrate present