Yeast & Cloning Sergio Peisajovich Lim Lab June 2007

Experimental Lab Why 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 Lab Yeast Life Cycle

Experimental Lab Yeast: Ideal Platform for Synthetic Biology

Parts/Devices/Modules are built in bacteria Empty initial plasmid Plasmid coding the desired device Transform into Yeast Experimental Lab Yeast: Adding parts… in plasmids

Experimental Lab Yeast: Adding parts… in plasmids growth in selective medium

Experimental Lab Yeast: Adding parts… in plasmids growth in selective medium

Experimental Lab Yeast: Adding parts… into the genome Homologous recombination allows genomic integration, but we still need to select:

Experimental Lab Yeast: 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* Homologous Recombination Yeast Chromosome Incoming Linear DNA URA3*URA3 Part/Device/Module Integration (Note that 2 copies, one defective and one functional, of the marker are generated) Yeast Chromosome

Experimental Lab Yeast: Adding parts… into the genome URA3 plasmid URA3 PCR product Linear DNA, ready for yeast transformation and integration yfg Homologous Recombination Yeast Chromosome URA3 Integration (yfg is now disrupted) Yeast Chromosome URA3

Part 1 plasmid Part 1 plasmid Part 2 Part 1 plasmid Part 2Part 3 Experimental Lab Combinatorial Cloning

A B B C C D A D AD Experimental Lab Combinatorial Cloning Based on Type IIs restriction enzymes

A B B C C D A D ADAD Combinatorial Cloning Experimental Lab Combinatorial Libraries

Experimental Lab Synthetic 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) PromTagEffectorZipperTerm

Experimental Lab Synthetic Biology as Engineering Engineering Negative Feedback Loops 1- Combinatorial Cloning in Bacteria 2- Transfer Constructs into Yeast 3- Analyze Pathway Behavior

Experimental Lab Synthetic Biology as Engineering Engineering Negative Feedback Loops 1- Combinatorial Cloning in Bacteria DONORSACCEPTORS

PromTagEffectorZipperTerm Experimental Lab Synthetic Biology as Engineering Engineering Negative Feedback Loops 1- Combinatorial Cloning in Bacteria

Experimental Lab Synthetic Biology as Engineering Engineering Negative Feedback Loops 2- Transfer Constructs into Yeast 3- Analyze Pathway Behavior FACS Microscopy