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What is Life?
Erwin Schrodinger
1944
Nobel Prize in Physics
1933
Atomic Theory
Max Delbruck
Nobel Prize in Medicine
1969
Seymour Benzer
rII……rapid lysis…. makes large plaques
E.coli
T4
K
B
WT
+
-
rII
-
+
Plate 0.1 ml
and count # plaques
103
106
109
Example-plate has 50 plaques, therefore 500 phage per ml at 109
or total = 5x 1011 per ml
Back to Benzer
E.coli
T4
K
B
WT
+
-
rII
-
+
Plate concentrated wild type on strain B to select rII mutants
Grow rII mutants to high density (strain B)
Plate onto strain K to select rare revertants
Some rII alleles revert (low frequency) others never revert
Two different non-revertable alleles and do mixed infection on
Strain K
Get very rare plaques that result from recombination
rII
rII-1
rII-2
Does not grow on K
Does not grow on K
Does not grow on K
Grows on K
Internal deletion
In the early days of phage genetics…..
Two types of mutants…
1. Altered plaque size and shape
2. Host range…..grow on certain strains of E. coli
Ultimately the goal became to identify every gene in the genome
Filling in the map with conditional mutants
Temperature sensitive mutants
Nonsense mutants
From the very beginning of Molecular Biology and Genetics
The goal has been to have a complete understanding of the genome
This means assigning a function to every gene in the genome
genotype
phenotype
DNA
Function
Assigning functions to a gene
When is it expressed?
Where is it expressed?
Is the protein modified?
Protein-Protein Interactions?
Phenotype when protein is reduced?
Phenotype when the protein is overexpressed?
GatewayTM Technology
Recombineering in vitro
A diversion to phage lambda
Life style choice…
Lysis vs lysogeny
Lysis-plaques
Lysogeny-phage infects the cell but is dormant
…The cell survives until there is some stress (uv light)
…Lysis
Lysogens (bacteria with dormant phage) are phage resistant
Lysogeny-Lysis
Lysis
attP
attB
Int
attR
attL
Lysogen
Stress
Xis
Lysis
Step 1…generate an “entry clone” with YFG
Step 2 recombine YFG into a destination vector
Destination vectors for every use
Assigning functions to a gene
When is it expressed? Microarray experiments
Where is it expressed? Epitope tagged protein
Is the protein modified? Gel shifts and mass spectrometry
Protein-Protein Interactions? GST or other affinity purifications
Phenotype when protein is reduced? siRNA
Phenotype when the protein is overexpressed? Strong promoter
But problems remain for tissue culture cells
Transfection (not transformation)
Stable (hard) or transient (easy)?
Transient:
Fraction of transfected cells is variable
Expression levels differs in individual cells
What cell types do you choose?
What is the isogenic wild type control?
HeLa cells karyotype from ATCC
Modal number of chromosomes= 82
Range = 70 to 164.
100% aneuploidy in 1385 cells examined.
Reducing expression
by shRNA
Libraries
Recall yeast one-step gene replacements
URA3
YFG1
URA3
in vitro approach
Use TAP purifications to
make protein chips
GST tagged protein kinases
Integrating Kinase
Expression Array
and TAP data