Week 12 Gene Expression Worksheet Fall 2020 PDF

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BIOS 310 Gene Expression Worksheet Student Name: _________________________

TA name: ____________________________

Lab Objectives  Introduce the fundamental concepts of gene expression and gene regulation.  Describe the processes of DNA transcription and translation.  Explore the techniques commonly used in gene expression. Lab Materials  S. marcescens bacterial culture starter plate.  LB agar plates and disposable loops. Background Information Gene regulation, a process of altering the expression of a gene, determines the functions of cells and how these cells respond and adapt to a constantly changing environment. For example, people with light skin get suntan when they are exposed to large amounts of sunlight. Intense sunlight causes an increase in the expression of the melanin gene, results in the production and accumulation of the melanin pigment. A suntan serves a function to protect the skin from the damaging effects of ultraviolet rays from the sun. In today’s lab, you will conduct a simple experiment to investigate the expression of the prodigiosin-producing genes in the Serratia marcescens bacteria. Prodigiosin is a red pigmented antibiotic produced by the Serratia species. The clinical interest on prodigiosin is enormous. This protein is reported to resist bacterial, fungal, protozoal, malarial, and cancer activities. The biosynthetic pathway of the prodigiosin in Serratia is a complex multi-step procedure that is beyond the scope of this course. However, students interested in this topic are encouraged to read Williamson et al. (2006), a Nature review paper entitled “The biosynthesis and regulation of bacterial prodiginines.” One of the many genes that control the production of prodigiosin in S. marcescens is a heatsensitive gene. At room temperature (~20–25C), the prodigiosin pigment is produced and the bacterial cells will appear red color. At higher temperature (>28C), this heat-sensitive gene will not express (it is turned off), prevents the completion of the prodigiosin synthesis pathway. The S. marcescens cells that grow in environment higher than 28C will appear white color because the prodigiosin red pigment cannot be fully synthesized. In this activity, you will examine the effect of temperature changes on the production of prodigiosin pigment by growing the S. marcescens cultures under different temperature treatments (room temperate and 37C). Note: Serratia may cause skin and eye irritations, please wear gloves and handle with care.

Temperature-Dependent Gene Expression Protocol

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BIOS 310 Gene Expression Worksheet 1. Students form groups of 4. 2. Each group receives the following items: LB agar plates x 4 3. Label the bottom of the plates as described below. Note: RT = room temperature. Plate 1 = “Group Name” – RT – 2 days Plate 2 = “Group Name” – 37C – 2 days Plate 3 = “Group Name” – RT day 1 / 37C day 2 Plate 4 = “Group Name” – 37C day 1 / RT day 2 4. Use the disposable loop, transfer a few S. marcescens colonies from the starter plate to inoculate Plate 1. The S. marcescens colonies on the starter plates will appear red color at room temperature. When you are transferring the colonies, make sure you obtain enough colonies with your loop, and distribute them evenly on your agar plates. When you complete this procedure, you should not see red bacterial colonies on your agar plate. 5. Discard the loop into the hazardous waste bag. Tape the plate to secure the lid. Don’t seal the plate completely because the bacteria need air to survive. 6. Repeat Step #4 and 5 to inoculate the remaining plates. 7. Place your plates upside down in the “completed plates” container. 8. TA will stack the plates (from different groups) with the same temperature treatments and tape them together. 9. The plates will be incubated in the following treatments: Plate 1 (RT – 2 days) and Plate 3 (RT day 1 / 37C day 2)  Leave them on the side bench in the designated area.  Incubate at room temperature for 1 or 2 days.  In day 2, prep TA or lab coordinator will transfer Plate #3 to the 37C incubator. Plate 2 (37C – 2 days) and Plate 4 (37C day 1 / RT day 2)  Incubate them in the 37C incubator for 1 or 2 days.  In day 2, prep TA or lab coordinator will transfer Plate #4 to side bench and incubate the plates at room temperature. 10. Predict the color of the S. marcescens colonies after the temperature treatment for each plate. Write down your expected results in Table 1. Show your answers to TA to receive points. TA will provide the answers to you in Gene Expression Lab II. 11. Before you leave the lab, please clean your bench with 70% ETOH and wash your hands with sanitizer.

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BIOS 310 Gene Expression Worksheet _________________________________________________________________________________________________________ Table 1. Expected and Observed Results of the Temperature-Dependent Gene Expression Experiment. Plat e

Temperature Treatment

1

RT – 2 days

2

37C – 2 days

3

RT day 1 / 37C day 2

4

37C day 1 / RT day 2

Expected Color

Observed Color

Temperature-Dependent Gene Expression Questions 1. What does it mean when a gene is “regulated” by temperature?

2. At what temperature did the prodigiosin-producing genes express in the S. marcescens culture? From the experiment you conducted in this lab, what evidence can you provide to support your claim?

3. What happened when the S. marcescens cultures were incubated at 37C for 1 day and then at room temperature for another day?

4. When the S. marcescens cultures were incubated at room temperature for 1 day before being transferred to 37C incubator, what happened to the prodigiosin synthesis pathway in the bacterial cells? A) All bacterial cells continue to produce red pigment.

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BIOS 310 Gene Expression Worksheet B) C) D)

All bacterial cells stopped producing red pigment. Pigment that had been made disappear. Bacterial cells slowly changed to white color. All bacterial cells stopped producing red pigment. Pigment that had been made remained. Bacterial cells that were previously exposed to room temperature continue to produce red pigment. New bacterial cells that were grown at 37C did not produce red pigment.

5. Imagine that you encounter two color morphs of S. marcescens in natural environment. If you do not have any prior knowledge on the prodigiosin synthesis pathway of these Serratia species, will you consider the red and white Serratia colonies as one species or two species? Do you think we should designate species based on morphological traits or genetic differentiation? Without limiting to the S. marcescens experiment, provide evidence and example to support your claim.

_________________________________________________________________________________________________________ Transcription and Translation Questions Note: Answer the following questions without taking start codon into consideration.

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BIOS 310 Gene Expression Worksheet 6. The template DNA sequence is 5’ ACCTGAGTC 3’, what is the transcribed mRNA sequence from 5’ to 3’?

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7. Refer to the table above, what are the amino acids that attach to the mRNA in Q6 from N-terminus to C-terminus?

8. When the second C in the template DNA sequence in Q6 mutates to T, mutated DNA is 5’ ACTTGAGTC 3’, describe the changes in the amino acids synthesis.

9. What are the three stop codons in RNA?

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BIOS 310 Gene Expression Worksheet 10. Identify the polypeptide that would be produced as a result of transcribing and translating the following DNA sequence from N-terminus to C-terminus. Template DNA: 3' GACTAAGCT 5' A) Leucine - Isoleucine - Arginine B) Methionine - Isoleucine - Arginine C) Stop D) Serine - Leucine - Valine E) Arginine - Leucine - Valine 11. What is the most common start codon in eukaryotes? Start codon: _____________ Amino acid: _____________________ 12. The strand of DNA used in Q10 problem undergoes a mutation, such that the first G from the 3' end is changed to a T. The mutant template DNA is: 3' TACTAAGCT 5'. What would change in the polypeptide? A) No change B) Polypeptide would be 1 amino acid shorter C) Polypeptide would be 1 amino acid longer D) 1 amino acid would substitute for another E) Translation would terminate within this stretch 13. The strand of DNA used in Q12 problem undergoes another mutation, this time the first G from the 5' end is changed to an A. The mutant template DNA is: 3' TACTAAACT 5'. What would change in the polypeptide? A) No change B) Polypeptide would be 1 amino acid shorter C) Polypeptide would be 1 amino acid longer D) 1 amino acid would substitute for another E) Transcription would terminate within this stretch 14. How many possible combinations of DNA sequences to code for a polypeptide consists of Glutamine – Proline – Cysteine?

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BIOS 310 Gene Expression Worksheet Real-time PCR Questions

15. Assuming the fluorescence threshold is set to be 1. Which gene has the highest gene expression rate? What is the Ct value? Gene with highest expression = ______________ Ct value = _______________

16. Which gene has the lowest gene expression rate? What is the Ct value? Gene with lowest expression = ______________ Ct value = _______________

17. Assuming real-time PCR was conducted on Gene 8, not shown on the graph above. If Gene 8 has a Ct value of 10, how would you interpret the result?

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