Cloning in Yeasts

5 Reasons to put DNA in eukaryotes

Make libraries

Learn about gene function

Make disease models

Change function

Produce proteins

4 Reasons to use Yeast

Libraries

Study gene function

Study cellular biology

Study proteins

Yeast reproduction

Asexual budding or sexual

Yeast organelles

Complete eukaryote set of nucleus, mitochondria, and secretion pathway

S. cerevisiae

Divides every 1.25-2 hours via budding

Single celled fungi with spherical cells

Can transform

6 Things Yeast models for

Vesicles

Cell Cycle

Signal transduction

proteasome

gene expression

morphology switch

Selection in yeast

auxotrophic strains that cannot make a certain amino or nucleic acid

Added plasmid will have a complementary copy of this gene so that transformed yeast can grow in a media without this component

YAC

have telomeres, cetromeres, selectable markers, origin of replication, large inserts, and two RE sites

EcoR1 in YAC

cut to add insert

BamH1 in YAC

cut to make DNA linear with insert in middle of two arms

how to prevent arms from reannealing

De-phosphorylate

Bases of yeast plasmids

2 microM circle

YIps

Integrative plasmids that always integrate into the yeast genome using homologous recombination

low efficiency but high stability

YEps

episomal plasmids that can integrate into or exit the host genome

can replicate autonomously and have high efficiency and low stability and a medium copy #

YRps

replicative plasmids that have an ARS and self-replicate

high copy number, efficency, low stability

YCps

Articificial chromosomes

1 copy, high efficiency, stable

Two reasons to use Yeast

Protein production and YAC library

P. Pasteris

yeast used for protein production due to having a better promoter

Basics of making proteins in yeast

Transform

lyse proteins to isolate

add signal sequence to secrete from cell

Adding a plasmid to a yeast

Clone in E coli

produce plasmids and shuttle to yeast

Two YIps components

bacterial plasmid backbone

complementary gene

YIp integration

Uses homologous recombination

Adds gene of interest + comp gene to host genome for selection

Deleting Yeast Genes

Do homologous recombination to add a complementary gene to the middle of a functional gene and see the effect

Southern Blotting or PCR to confirm

3 Major Yeast Studies Accomplishments

1. 6000 deletion mutant strains made

2. All genes tagged with GFP

3. Protein interactions discovered

Tagging with GFP

homologous recombination used to add GFP gene and selectable marker next to protein coding region

Why is P. pastoris used over S. cerevisiae sometimes?

stronger promoter

more accurate glycosylation

Yeast and toxin detection

Add GFP to promoter only activated if DNA is damaged and add sample

No effect = nontoxic

reduction in cell concentration = toxic

cells turn green = mutation in promoter = genotoxic