Genetics Lab report

BIO 224 L. Hollis-Brown

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LAB 11: GENETIC SCREEN OF MODIFIED YEAST OBJECTIVES 1. Perform a genetic screen on fission yeast transformed with modified pMZ379. 2. Explain the purpose of the genetic screen in the context of CRISPR/Cas gene

editing in fission yeast. BEFORE LAB 1. Read the lab handout in its entirety. If you do not prepare adequately for lab,

you will not be able to complete the lab in the time allotted. 2. Complete the pre-lab quiz before lab.

LAB SAFETY 1. Wear gloves and safety glasses throughout the procedure. 2. Clean your workstation before and after the procedure. 3. Wear closed-toed shoes and secure loose hair and clothing. 4. Dispose of reagents and used materials in labeled containers only. 5. Wash your hands after the procedure, even if you have been wearing gloves.

GENERAL NOTES ON LAB 1. Be sure to ask questions if you are unsure of any instructions.

2. Take careful notes as you do the lab. You will need these notes to write your final paper at the end of the semester.

3. You will turn in this lab handout for a grade. Be sure you have all questions answered in the handout.

INTRODUCTION In the previous lab, you transformed competent Schizosaccharomyces pombe with pMZ379. The modified plasmid contained: the sgRNAs previously inserted by PCR, and a selectable marker, the nourseothricin resistance gene (NAT: nourseothricin acetyl transferase). After transforming the yeast, you grew them on agar containing nourseothricin. The yeast that took up the plasmid were able to express the NAT gene and were able to grow in the presence of nourseothricin, while the yeast that were not transformed did not contain the plasmid and did not have resistance to the antibiotic. In this week’s lab, you will do a genetic screen, which is a process to identify individual cells that have a particular phenotype. In this experiment, the yeast cells that the plasmid with the inserted sgRNA will be able to silence the target gene. You will transfer the yeast from the nourseothricin plates on to other selective media plates, which will allow only the yeast with the phenotype of interest to grow.

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Questions

1. Review the Lab 5 handout (CRISPR Overview and Bioinformatics), in which you identified the phenotype of the yeast cells with the silenced genes. Fill in the table below with the genes your group was assigned. For each gene, write the predicted phenotype of the cells with the silenced gene, and the type of media you would need to grow the cells on in order to determine if the gene is silenced.

Gene Name Expected Phenotype of Cells

With Silenced Gene Type of Media Needed To

Screen Cells

Table 1. Selective media needed for genetic screen.

2. Work with your group to determine the control treatments that you will use in your experiment. Identify these controls, and describe the specific purpose of each control below.

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GENETIC SCREEN OF MODIFIED S. pombe CELLS You will selectively transfer yeast cells from the nourseothricin agar plates onto selective media plates that you identified in the table above. In this procedure, you will use a grid pattern to transfer cells using sterile toothpicks.

It is very important that you transfer the smallest yeast colonies that appear on the agar plates. This is because the Cas9 enzyme is detrimental to the yeast cells. The colonies that grow the most slowly are most likely to be composed of cells that have a functioning, non-mutated Cas9 gene. Materials Per Group

yeast on nourseothricin agar plates from previous lab 40 sterile toothpicks in capped test tubes 1 test tube rack 4 paper grids 1 250 mL waste beaker for toothpicks 1 bottle 95% ethanol 2 fine Sharpies Shared by Class 1 box ea. non-latex gloves, small, medium, large safety glasses selective media plates:

• YES5 + LiCl • YES5 + 15mM caffeine • synthetic medium without adenine • YES5

Procedure for Genetic Screen 1. Wear gloves and safety glasses.

2. Disinfect your workstation with 95% ethanol.

3. You should take a photo of the results of the yeast growing on agar + nourseothricin for your paper. If you do not have a phone or camera with which to take a photo, let your instructor know.

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4. Obtain selective media plates for each gene. Remember to include a plate for the experimental treatment, and one or more plates for the controls. Label each plate with:

• your initials, • the date, • the gene name, • contents of the plate, organism, and treatment.

5. On the nourseothricin agar plates from last week, label the smallest colonies

with a marker (on the plastic side of the plate, beneath the agar), from 1 to at least 10. If you can identify more than 10 small colonies, then you should. If you do not have at least 10 colonies, then label all colonies on your plate.

6. For the your gene, obtain the media plates that you will need for your experimental treatment and controls.

7. For each plate, on the side with the agar, make a short line with a marker on the plastic from the edge of the agar plate toward the center of the plate. Then, place the paper grid beneath the agar plate, so that the lines on the grid and the plate match up. This line helps you keep your plate and grid lined up if one of them gets moved.

Figure 1. Grid for agar plate.

8. Line up the plates in a row, with the source plate (from which you are taking the colonies) next to the receiving plates (where you are placing the colonies: experimentals and controls).

9. Obtain a sterile toothpick from the test tube. Touch only one end of the toothpick with your fingers. If you accidentally touch the other end of the toothpick, discard it without using it.

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10. On the nourseothricin plate, identify the colony you numbered “1”.

11. Use the sterile toothpick to gently scrape the colony of cells numbered “1” from the nourseothricin plate (the source plate).

12. In the square numbered “1” on the grid of your first receiving plate, gently touch the end of the toothpick and wipe it in this square. You will not see the cells on the plate with the naked eye. They will need to grow into colonies over the next few days to become visible.

13. Throw away the toothpick in the marked container.

14. Record the origin of Colony 1 for that plate in the table on the last page. Be sure to label the information you put in the chart!

15. Repeat Steps 9 through 15 for the remaining numbered colonies. You may have more than one source plate, and more than one receiving plate, depending on the controls that you are running.

16. Repeat Steps 5 through 16 for the second gene.

17. The plates will be placed in an incubator for several days, and can be checked for growth in the following lab.

Questions

1. Explain why it was important to transfer the smallest colonies of yeast to the selective media plates.

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2. If the yeast do not grow on your selective media plates, describe at least two different errors that may have occurred. Do not just identify the errors; describe how they would affect your results. Choose errors that are likely to have occurred.

POST-LAB 1. Be sure all toothpicks are disposed of in the waste beakers. 2. Dispose of yeast agar plates in biohazard containers at the back of the room. 3. Wipe down your workstation with ethanol or Lysol. 4. Throw away all gloves and paper towels. 5. Wash your hands. 6. Make sure that all materials are clean and returned to your kit. 7. You will turn in this handout for a grade. Be sure to answer all questions in

the handout.

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Table 2. Origin and placement of yeast colonies from source to receiving plates.