Lab 22

Question 1

Focus lab 22

Answer 1

Use CRISPR-Cas9 system to make a partial deletion of the RFP gene in yeast strain RJY29

Question 2

Model Organism lab 22

Answer 2

Sacchoromyces cerevisiae - single celled eukaryote (10 microns)

Question 3

Why Saccharomyces cerevisiae?

Answer 3

Cheap and easy to grow, short generation time (90 min), can be stored frozen, grows in haploid or fiploid state, exists in two sexes for genetic mating studies, many mutant strains available, deletions of nearly every gene available

Question 4

Saccharomyces cerevisiae genome length and chromosome #

Answer 4

12 Mb and 16 chromosomes

Question 5

Components of CRISPR-Cas9

Answer 5

1) sgRNA binding to Cas9, 2) PAM sequence, 3) Cas9 protein, 4) Matching DNA target sequence

Question 6

What is the purpose of the HF?

Answer 6

Once CRISPR-Cas9 creates double stranded break, yeast cell repairs the break by non homologous end joining or homology directed repair with HF

Question 7

what is sgRNA

Answer 7

the two strands of DNA typically used in bacterial CRISPR fused together by linker into single guide RNA strand

Question 8

Difference between NHEJ and HDR

Answer 8

NHEJ does not require a template strand and results in two strands of different size (by insertions and deletions). HRD requires donor DNA and has sequences the same as the ends of the cuts with stuff between. HDR requires HF but lowers instance of error and causes uniformity of size

Question 9

What do the RFP and LEU2 gene do

Answer 9

Causes red fluorescence and makes cells able to produce amino acid leucine

Question 10

Both the RJY29 and SEY6210 cells are ______

Answer 10

Leu2-

Question 11

pND496 has ______ which pND434 does not. However, both cells have ________

Answer 11

gRFP2 gene. Cas9 gene and LEU2 gene

Question 12

What does salt (LiOAc) do in transformation process

Answer 12

makes cells competent for transformation, increase cell wall permeability (leaky cell wall)

Question 13

Why heat shock after adding transformation mix?

Answer 13

increase entry of DNA into cell

Question 14

What does PEG do

Answer 14

Deposit DNA onto cell membrane - forms PEG shell which changes osmotic pressure and makes cell leakier, adsorb DNA and ssDNA onto cell

Question 15

What does ssDNA do

Answer 15

saturate DNA binding sites on cell wall - without ssDNA most plasmid DNA will stick to cell wall

Question 16

Why grow cells to the log phase before transformation?

Answer 16

Optimal density and cellular state for DNA uptake