Download Yeast - iGEM 2007

Yeast & Cloning
Sergio Peisajovich
Lim Lab June 2007
Why Yeast?
Experimental Lab
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).
Yeast Life Cycle
Experimental Lab
Yeast: Ideal Platform for
Synthetic Biology
Experimental Lab
Add parts, devices or even modules (in an
ТextragenomicУ format -plasmid -based- or Тinte gratingУ them
within the yeast genome.
Delete specific yeast genes, to r emove Тbackground У or
interference.
Add Тreporter genesУto monitor in real time the function of
the synthetic parts/devices/modul es unde r study.
Life cycle fa st enough so that we could do all these
genetic manipul ations in a reasonable amount of time.
Yeast: Adding parts…
in plasmids
Experimental Lab
Parts/Devices/Modules are built in bacteria
Transform into
Yeast
Empty initial
plasmid
Plasmid coding
the desired
device
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
plasmid
Experimental Lab
Part/Device/Module
plasmid
URA3
Digest with specific
restriction enzyme
Linear DNA, ready for yeast
transformation and integration
Part/Device/Module
Incoming
Linear DNA
Homologous
Recombination
URA3*
Yeast
Chromosome
Integration
(Note that 2 copies, one defective
and one functional, of the marker
are generated)
URA3*
Part/Device/Module
Yeast
Chromosome
URA3
Yeast: Adding parts…
into the genome
Experimental Lab
PCR product
URA3
plasmid
URA3
Linear DNA, ready for
yeast transformation
and integration
URA3
Integration
(yfg is now disrupted)
yfg
URA3
Homologous
Recombination
Yeast
Chromosome
Yeast
Chromosome
Experimental Lab
Combinatorial
Cloning
Part 1
Part 1
Part 2
plasmid
plasmid
Part 1
Part 2
plasmid
plasmid
plasmid
plasmid
Part 3
Experimental Lab
Combinatorial
Cloning
A
B
B
C
A
D
C
D
Based on Type IIs
restriction enzymes
A
D
Experimental Lab
Combinatorial
Cloning
A
B
B
C
A
D
C
D
Combinatorial
Libraries
A
D
A
D
Experimental Lab
Synthetic Biology as
Engineering
Engineering Negative
Feedback Loops
Prom
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)
Tag
Effector
Zipper
Term
Synthetic Biology as
Engineering
Engineering Negative
Feedback Loops
Experimental Lab
1- Combinatorial Cloning in Bacteria
2- Transfer Constructs into Yeast
3- Analyze Pathway Behavior
Synthetic Biology as
Engineering
Engineering Negative
Feedback Loops
Experimental Lab
1- Combinatorial Cloning in Bacteria
DONORS
ACCEPTORS
Experimental Lab
Synthetic Biology as
Engineering
Engineering Negative
Feedback Loops
1- Combinatorial Cloning in Bacteria
Prom
Tag
Effector
Zipper
Term
Synthetic Biology as
Engineering
Engineering Negative
Feedback Loops
2- Transfer Constructs into Yeast
3- Analyze Pathway Behavior
FACS
Microscopy
Experimental Lab