Erwin Schrodinger

What is Life?

1944

Nobel Prize in Physics 1933Atomic Theory

Max Delbruck

Nobel Prize in Medicine 1969

rII……rapid lysis…. makes large plaques

Seymour Benzer

rII

WT

K B

- +

+ -

E.coliT4

103 106 109

Plate 0.1 ml and count # plaques

Example-plate has 50 plaques, therefore 500 phage per ml at 109

or total = 5x 1011 per ml

Back to Benzer

rII

WT

K B

- +

+ -

E.coliT4

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 onStrain K

Get very rare plaques that result from recombination

rII

rII-1

rII-2

Internal deletion

Does not grow on K

Does not grow on K

Does not grow on K

Grows on K

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 TechnologyRecombineering 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

Lysogen

attP

attB

attL attR

Int

Xis

Lysis

Lysis

Stress

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?

Modal number of chromosomes= 82

Range = 70 to 164. 100% aneuploidy in 1385 cells examined.

HeLa cells karyotype from ATCC

Reducing expression by shRNA

Libraries

Recall yeast one-step gene replacements

YFG1

URA3

URA3

in vitro approachUse TAP purifications tomake protein chips

GST tagged protein kinases

Integrating KinaseExpression Arrayand TAP data