Mol Gen Lab 7 Assignment (lac Operon assignment) PDF

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Lab 7 Assignment 26 Marks Worth 3% of Final Course Grade

NOTE on symbolism. When prokaryotic genotypes and phenotypes are being discussed the convention is the following: Genotypes are always written in italics using lower case letters (3 letters in length; underlining can substitute for italics in the case of hand written information). With respect to operon genotypes, only the fully wild type genotype is designated as lac+. Phenotypes are written without italics (lac+ or lac-) and refer to the ability of the organism to do a specific metabolic task (in this case use lactose as an energy source) and do not necessarily mean that the genotype would also be lac+ just because the phenotype is lac+. 1) What is the name and function of the following region of the lac operon: lacZ+ (1 mark) Repression gene- codes for a repressor protein which binds to the operator region thus turns off the entire operon in the absence of lactose. 2) What is the name and function of the following region of the lac operon: lacY+ (1 mark) Operator gene- binding site of the repressor protein when the repressor is bound to the operator, RNA polymerase is unable to transcribe the genes of the operon. When the lactose is present in the environment, it will bind to the repressor protein, thus inactivating and preventing it from binding to the operator. 3) Define positive regulation and negative regulation as it applies to prokaryotic gene regulation. Explain what type of regulation is seen in the lac operon? (3 marks)

Positive regulation is the binding of a specific protein (activator) is needed for transcription to occur. DNA bound activators are able to regulate transcription by helping with ignition. In order to do so however, they sometimes will tether RNA polymerase to the promoter. Negative gene regulation is the binding of a specific protein (repressor) that will inhibit transcription from occurring. DNA bound repressors will act as means to prevent RNA polymerase from binding to the promoter, or by blocking the movement of RNA polymerase. This type of regulation can be observed in the lac operon.

4) Define catabolite repression. Fully describe how it affects the lac operon? (3 marks)

catabolite repression. It is positive control of the lac operon. The effect will be an increase in the rate of transcription as a regulatory protein which will increase (upregulation) the rate of transcription of an operon. Glucose can inhibit the expression of the genes of lac operon when it is present. It will prevent expression of lactose utilizing genes. While in the presence of glucose, cAMP levels will decrease and it won’t be able to interact with CAP and cAMP – CAP complex will fail to bind to the promoter region of lac operon and as a result RNA polymerase will not be able to transcribe lac utilizing genes and the lactose operon as a result will remain in an inactive state.

5) Observe the colour of growth (white or green) for Strains A-D on EMB within the Lab 7 data file to fill in Table 1 below. (2 marks) Table 1: Strain Colour of Growth on Phenotypically lac+ OR EMB lac- ? A White Z–Y–IB Dark Green Z + Y+ I + C White Z–Y–ID Dark Green Z + Y+ I + 6) Explain what being phenotypically lac+ versus lac- means. (1 mark) PHENOTYPICALLY lac + means it can utilize lactose as an energy source and will as a result grow green on the EMB. Phenotypically lac – means it is unable to utilize lactose and will grow white 7) If you had assayed for the enzyme during Step 2 of the lac operon exercise, this is what you would have expected to see in each of your 24 sample tubes. Table 2: B-Galactosidase activity assayed with ONPG in all four strains under various conditions, which gave different colours of a yellow gradient, such as colourless (C), pale yellow (PY), yellow (Y) and bright yellow (BY)

Time (min)

Strain A

Strain A

Strain B

Strain B

Strain C

Strain C

Strain D

Strain D

With IPTG

With Water

With IPTG

With Water

With IPTG

With Water

With IPTG

With Water

0

C

C

Y

Y

C

C

C

C

20

C

C

BY

BY

PY

C

PY

C

40

C

C

BY

BY

Y

C

Y

C

Table 3: B-Galactosidase activity assayed with ONPG in all four strains under various conditions which gave different absorbance reading at 420 nm Time (min)

Strain A

Strain A

Strain B

Strain B

Strain C

Strain C

Strain D

Strain D

With IPTG

With Water

With IPTG

With Water

With IPTG

With Water

With IPTG

With Water

0

0.023

0.013

0.483

0.385

0.023

0.015

0.013

0.013

20

0.025

0.015

0.613

0.662

0.135

0.022

0.136

0.014

40

0.025

0.018

0.810

0.813

0.209

0.030

0.335

0.023

Graph the Absorbance420 against the induction time for all four strains, with and without the inducer IPTG (from table 3 above). Graph all 8 lines on the same graph because you need to be able to compare the induction profiles of the 4 strains. Make sure to label your axes and include a figure number and descriptive title below the graph. Also include a legend to distinguish between the 8 lines on your graph. (5 marks) Note: You may use regular graph paper for the graph, take a photo, then paste into your assignment OR make the graph in excel then copy into your assignment– your choice! 0.9 0.8

Absorbance (420nm)

0.7 Strain A "IPTG"

0.6

Strain A "Water" 0.5

Strain B "IPTG" Strain B "Water"

0.4

Strain C "IPTG" 0.3

Strain C "Water" Strain D "IPTG"

0.2 Strain D "Water"

0.1 0 0

10

20

30

40

50

Time (min)

Figure 1. Absorbance at 420nm of the 4 bacteria strains over the course of 40 minutes in either water or IPTG.

8) Fully explain how a higher absorbance reading at 420 nm can be related to more β-galactosidase activity in the sample. (2 marks) A higher absorbance reading at 420 nm can be related to more B-galactosidase activity because whenever there is an increase in the activity of B-galactosidase the absorbance has also shown to get higher. The same can go for when there is a decrease in B-galactosidase the absorbance will also drop, they are directly proportional to each other, a change in activity will either increase/decrease absorbance depending on increase/decrease observed in Bgalactosidase.

9) Examine the IPTG induction results (from tables 2 and 3 in question 7) and correlate these with the EMB lactose agar results (from question 5). Assign one of the four possible genotypes to each of the strains A, B, C, and D. Support the genotype assignments that you have made. Explain how each genotype produces both the IPTG and water induction characteristics and the observed phenotype on EMB agar. (8 marks) Table 4. The various genotypes assigned to the four strains A, B, C, D. Strain Genotype lac- Z- Y+ I + A lac- Z+ Y+ IB lac- Z+ Y- I+ C lac+ Z+ Y+ Z+ D Strain A has been assigned the genotype lac- Z- Y+ I +, this is the result of the strain never making any enzyme as we can observe from both the color of the IPTG samples and water samples being clear. This indicates that there was not enzymatic activity present thus, nothing can be observed. A lack of activ ity is most likely the result that Z is (-), meaning it is off. Z is a structural gene for Bgalactosidase, so if Z is “off”, nothing is going to be observed as a result of Bgalactosidase not being hydrolyzed. Strain B has been assigned the genotypes lac- Z+ Y+ I-, this is the result of this strain making a continuous amount of enzyme regardless if it is in water or IPTG. What can be determined is that lactose is being hydrolyzed to glucose + galactose. High absorbance readings are also strong indicators of activity taking place. I- is off, which indicates this is the repressor that will code for repressor proteins. Which will bind to the operator and turn it off. Thus what the experimenter needs to do is induce the operon, to make it move the repressor protein it will need to be strongly induced with a strong inducer. Based on results IPTG was not strong enough however, water was so IPTG seems to be the control in this case because water was the stronger inducer. But, in our case the repressor is off so nothing is binding to the operator which is why B-galactosidase was working in both conditions. Strain C has been assigned the genotype lac- Z+ Y- I+, this is the result of no enzyme being made in water and having a clear color, as well as having a very low absorbance reading. However, in IPTG we do observe the yellow color, as well as a much high absorbance reading. This strain appears to have a nonfunctioning Y which is a structural gene for B-galactosidase permease, as a result of its absence B-galactosidase will be restricted to inside the side, while lactose is stuck outside, since permease is needed for the transfer of lactose across the membrane however, it is important to note that we are able to observe some form

of activity most likely the result of the presence of lactose that was already in the cell beforehand. Strain D has been assigned the lac+ Z+ Y+ Z+, Z is working so we know enzymes are being made, Y+ is on so lactose is able to move across membrane, however, as a result of I+ the operon will be left off as a result of the repressor protein being bound to it. So what is needed is a strong inducer to remove the repressor, which can be done with IPTG, which means water will be clear in color and show no activity which is exactly what has been observed....