Yeast & Cloning

1. Yeast & Cloning Sergio Peisajovich Lim Lab June 2007

2. 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).

3. Experimental Lab Yeast Life Cycle

4. Experimental Lab Yeast: Ideal Platform for Synthetic Biology

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

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

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

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

9. Experimental Lab Yeast: Adding parts… into the genome Part/Device/Module Part/Device/Module plasmid plasmid URA3 Digest with specific restriction enzyme Linear DNA, ready for yeast transformation and integration Part/Device/Module Incoming Linear DNA Integration (Note that 2 copies, one defective and one functional, of the marker are generated) Homologous Recombination URA3* URA3* Part/Device/Module URA3 Yeast Chromosome Yeast Chromosome

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

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

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

13. Experimental Lab B A C B D C D A A D A D Combinatorial Cloning Combinatorial Libraries

15. Experimental Lab Synthetic Biology as Engineering Engineering Negative Feedback Loops Prom Tag Effector Zipper Term 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)

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

17. Experimental Lab Synthetic Biology as Engineering Engineering Negative Feedback Loops 1- Combinatorial Cloning in Bacteria DONORS ACCEPTORS

18. Experimental Lab Synthetic Biology as Engineering Engineering Negative Feedback Loops 1- Combinatorial Cloning in Bacteria Prom Tag Effector Zipper Term

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