Genetics Lab report

BIO 224 Lisa Hollis-Brown

1 | 8

LAB 10: TRANSFORMATION OF COMPETENT YEAST CELLS WITH MODIFIED PLASMID

OBJECTIVES 1. Perform a transformation of competent Schizosaccharomyces pombe with modified

pMZ379. 2. State the ploidy of S. pombe, and explain the relationship of the stages of the

eukaryotic cell cycle to the process of CRISPR/Cas gene editing in the yeast. 3. Explain the purpose of the transformation of S. pombe with the modified plasmid 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. 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.

BIO 224 Lisa Hollis-Brown

2 | 8

INTRODUCTION You previously learned that genetic transformation is the uptake and expression of exogenous DNA by a cell. Transformation is most often observed and described in bacterial and archaeal cells (see van Wolferen, et. al., 2016). However, some eukaryotic organisms, such as yeasts (Kawai, 2010) and certain plants (Gelvin, 2003) are able to be transformed with plasmids. The transformation of eukaryotic cells poses a few extra challenges compared to the transformation of bacterial cells. First, the exogenous DNA must not only pass through the cell wall and membrane, but must also be imported into the nucleus of the cell. In addition, transformation success can be decreased due to cells having multiple copies of chromosomes, or having duplicated chromosomes. Before continuing your reading, review some of the concepts from earlier in the semester regarding the eukaryotic cell cycle and mitosis. Questions (Answer these before coming to lab.)

1. Review the stages of the eukaryotic cell cycle. Suppose a 2N= 4 cell goes through interphase. What are the number and structure of the chromosomes during:

• G1 phase?

• S phase?

• G2 phase?

2. If an N= 3 cell undergoes mitosis, what will be the number and structure of the chromosomes the start of mitosis? What will the number and structure of the chromosomes be at the end of mitosis and cytokinesis?

BIO 224 Lisa Hollis-Brown

3 | 8

Many eukaryotic cells have multiple copies of each chromosome (i.e., are diploid or polyploid.) So, even if exogenous DNA is taken up by the cell and nucleus, and it is inserted into a chromosome through homologous recombination, the expression of the exogenous DNA may be masked by alleles on the homologous chromosome. Finally, if a cell is in S phase or G2, the exogenous DNA may be inserted into only one of the chromatids. If the remaining chromatids do not contain the new DNA, this reduces the likelihood of creating daughter cells through mitosis that contain the newly inserted DNA. In addition, a dividing eukaryotic cell may revert the newly modified DNA back to its original form, based on the original template from the sister chromatid.

In the case of the transformation of the S. pombe in this lab, the goal is not to insert a new gene into the genome of the yeast. The goals are: 1.) to allow the yeast to take up the plasmid to express the sgRNA gene, Cas9 gene, and NAT gene; and 2.) to silence a target gene on a yeast chromosome.

Questions 1. In your own words, describe three different, specific challenges to genetic

transformation in eukaryotic cells.

TRANSFORMATION OF COMPETENT S. pombe CELLS Schizosaccharomyces pombe are called fission yeast, because they elongate during cell division and divide into equal-sized cells during cytokinesis (as opposed to creating the small buds found in Saccharomyces cerevisiae.) S. pombe have a total of three chromosomes, and exist primarily in the haploid condition. The yeast cells that you will transform today have been arrested in the G1 phase of the cell cycle. They will continue through the cell cycle after they have been transformed.

BIO 224 Lisa Hollis-Brown

4 | 8

In this lab, you will transform S. pombe cells with pMZ379. This plasmid contains the sgRNA genes that you inserted and amplified during PCR. You then isolated the modified, circularized plasmid from bacteria. Recall that the plasmid also has a nourseothricin resistance gene (NAT: nourseothricin acetyltransferase), which allows the transformed yeast to grow in the presence of the antibiotic nourseothricin. In order to determine if the yeast have taken up the plasmid, the yeast that you transform today will be grown on agar plates containing nourseothricin.

In the next lab, you will do a genetic screen, by growing the yeast that survive in the nourseothricin environment on agar plates that will allow you to detect if the target gene has been silenced. Recall that the plasmid contains the sgRNA gene that will be transcribed into a single guide RNA (sgRNA). The Cas9 gene on the plasmid will be transcribed and translated into the Cas9 endonuclease. Ultimately, the Cas9 enzyme will carry the sgRNA to the target gene (avt5, ade2, and/or trr1), detect the PAM site on the yeast DNA, and then cut the target gene. When the cell tries to repair the break in the DNA, it may ultimately make errors and silence the gene. Cells grown in environments such as high salt, high caffeine, or absence of adenine will allow you to determine if the corresponding genes have been silenced.

Questions

1. What is the ploidy and equation for a typical S. pombe cell?

2. Describe specifically how the ploidy of the yeast cells, and the phase in which the cells have been arrested, will increase the success of creating cells that will express the phenotype of the silenced target gene.

BIO 224 Lisa Hollis-Brown

5 | 8

3. Work with your group to determine what your experimental and control treatments should be for this step of the experiment. Review these with your instructor, and then tell the instructor which materials you will need. Clearly describe your control and experimental treatments below. Explain the specific purpose of each treatment.

BIO 224 Lisa Hollis-Brown

6 | 8

Materials

Per Group 1 each purified, modified plasmids from plasmid recovery lab (stored in freezer) 3 aliquots competent yeast in G1 phase (stored in freezer) 1 mL 50% PEG4000 6 mL EMM-N broth 1 set 2-20, 10-100, and 100-1000 μL pipettors 1 each box of sterile large and small pipette tips 1 bag sterile microfuge tubes 2 foam microfuge tube racks 1 foam cooler 1 250 mL waste beaker for tubes and tips 1 bottle 95% ethanol and DNAase Away for disinfecting 2 fine Sharpies Shared by Class 3 aliquots herring sperm DNA 2 microcentrifuges 1 ice bucket with ice 1 box ea. non-latex gloves, small, medium, large safety glasses 2 hot blocks YES5 + Nourseothricin agar plates YES5 agar plates

Procedure for Yeast Transformation 1. Wear gloves and safety glasses.

2. Disinfect your workstation and instruments with DNAase Away and ethanol.

3. Keep all materials on ice.

4. You will need one tube of competent yeast for each of your treatments (experimentals and controls). Thaw your aliquots of competent yeast cells for 2 min in a 40OC heat block.

5. Label the tubes of yeast as experimental tubes "+", and control tubes "0”, and the gene name. Also include your group’s initials. If you are running more than one control, be sure to indicate the contents of each control.

BIO 224 Lisa Hollis-Brown

7 | 8

6. To all of the tubes, add 2 µL of denatured herring sperm DNA. The herring ssDNA acts as a carrier of the plasmid, and helps the plasmid enter the yeast cells. The aliquots of herring sperm DNA are in an ice bucket at the front of the room. Bring the tube of herring DNA to your workstation, on ice, and then return it to the front of the room when you are finished.

7. To your experimental tubes of yeast add 10 µL of recovered plasmid from your plasmid recovery tubes.

8. To all tubes, add 145 µL of 50% PEG4000, and mix well by inverting the tube 4 to 5 times.

9. Incubate the tubes in a hot block for at least 15 min at 43 °C. This step is crucial for allowing the plasmid to get past the chitinous cell wall of the yeast.

10. Centrifuge all tubes for 3 min at 1600 g at room temperature.

11. Carefully remove the supernatant, leaving a pellet with yeast cells. Gently pour some of the supernatant out, and then pipette out the remaining supernatant. Do not disturb the pellet. The yeast pellets tend to disintegrate rapidly. If your pellet of cells seems to be re-dissolving into the supernatant, then centrifuge them again and remove the remaining supernatant. Make sure that all of your tubes have about the same amount of liquid in them before centrifuging.

12. Resuspend the pellet of yeast cells in 1 mL EMM-N, by gently pipetting the cells up and down.

13. Place the cells in a plastic microfuge tube rack at room temperature. The cells will incubate for 16 hours (or overnight) at room temperature. This allows the cells to reproduce, creating many new cells.

14. The following day, your instructor will:

a. Centrifuge cells for 3 min at 1600 g at room temp. Cells will be in a pellet at the bottom of the tube.

b. Remove 700 µL of supernatant from experimental tube, and 900 µL from control tube, and discard.

c. Resuspend cells in remaining liquid (300 µL experimental, 100 µL control).

d. Plate cells on YES + Nourseothricin media plates.

e. Incubate cells at 32OC for four to eight days.

15. You will use the colonies on these plates in the following lab to do a genetic screen.

BIO 224 Lisa Hollis-Brown

8 | 8

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

the handout. REFERENCES Gelvin, S.B. 2003. Agrobacterium-mediated plant transformation: the biology behind

the “gene-jockeying” tool. Microbiol Mol Biol Rev 67(1):16-37.

Kawai, S., Hashimoto, W., and Murata. K. 2010. Transformation of Saccharomyces cerevisiae and other fungi. Bioeng Bugs 1(6):395-403.

van Wolferen, M., Wagner, A., van der Does, C., and Albers, S. 2016. The archaeal Ced system imports DNA. Proc Nat Acad Sci 113(9):2496-2501.