Yeast & Cloning Sergio Peisajovich Lim Lab June 2007.

Yeast & Cloning

Sergio Peisajovich

Lim Lab June 2007

Experimental LabWhy Yeast?

The yeast Saccharomyces cerevisiae (also called “baker’s yeast”) is probably the ideal eukaryotic microorganism for biological studies.

Yeast genome: fully sequenced and easy to manipulate.

Basic mechanisms of yeast cell biology (such as DNA replication, recombination, cell division and metabolism) are highly similar to that of higher organisms (including humans).

Experimental LabYeast Life Cycle

Experimental LabYeast: Ideal Platform for Synthetic Biology

Add parts, devices or even modules (in an “extra-genomic” format -plasmid-based- or “inte grating” them within the yeast genome. Delete specific yeast genes, to r emove “background” or interference. Add “reporter genes” to monitor in real time the function of the synthetic parts/devices/modules under study.

Life cycle fa st enough so that we could do all these genetic manipulations in a reasonable amount of time.

Parts/Devices/Modules are built in bacteria

Empty initial plasmid

Plasmid coding the desired

device

Transform into Yeast

Experimental LabYeast: Adding parts… in plasmids

Experimental LabYeast: Adding parts… in plasmids

growth in selective medium

Experimental LabYeast: Adding parts… in plasmids

growth in selective medium

Experimental LabYeast: Adding parts… into the genome

Homologous recombination allows genomic integration, but we still need to select:

Experimental LabYeast: Adding parts… into the genome

Part/Device/Module

URA3

plasmid

Digest with specific restriction enzyme

Part/Device/Module

plasmid

Linear DNA, ready for yeast transformation and integration

Part/Device/Module

URA3*

HomologousRecombination

YeastChromosome

Incoming Linear DNA

URA3* URA3Part/Device/Module

Integration(Note that 2 copies, one defective and one functional, of the marker

are generated)

YeastChromosome

Experimental LabYeast: Adding parts… into the genome

URA3

plasmid

URA3

PCR product

Linear DNA, ready for yeast transformation

and integration

yfg

HomologousRecombination

YeastChromosome

URA3

Integration(yfg is now disrupted)

YeastChromosome

URA3

Part 1

plasmidplasmid

Part 1

plasmidplasmid

Part 2

Part 1

plasmidplasmid

Part 2 Part 3

Experimental LabCombinatorial Cloning

A B B C C D

A D

A D

Experimental LabCombinatorial Cloning

Based on Type IIsrestriction enzymes

A B B C C D

A D

A D A D

Combinatorial Cloning

Experimental Lab

CombinatorialLibraries

Experimental LabSynthetic Biology as Engineering Engineering Negative

Feedback Loops

Negative Effectors to be used:OspF (MAPK Phosphothreonine Lyase) YopJ (MAPKK Ser/Thr acetylase)YopH (MAPK Tyr phosphatase)

Promoters to be used:Constitutive expression (Adhp, CycIp, Ste5p)Inducible by pathway activation (STLp, Fig1p)

Protein-interaction domains:Leucine Zippers (high and medium affinities,some with degradation motif)

Prom Tag Effector Zipper Term

Experimental LabSynthetic Biology as Engineering Engineering Negative

Feedback Loops

1- Combinatorial Cloning in Bacteria

2- Transfer Constructs into Yeast

3- Analyze Pathway Behavior

Experimental LabSynthetic Biology as Engineering Engineering Negative

Feedback Loops

1- Combinatorial Cloning in Bacteria

DONORS ACCEPTORS

Prom Tag Effector Zipper Term

Experimental LabSynthetic Biology as Engineering Engineering Negative

Feedback Loops

1- Combinatorial Cloning in Bacteria

Experimental LabSynthetic Biology as Engineering Engineering Negative

Feedback Loops

2- Transfer Constructs into Yeast

3- Analyze Pathway Behavior

FACS

Microscopy