Transcript Lecture 11 - Instructional Web Server

Lecture 11
• Assignment 4
• Notebook
• Beta-galactosidase assay
• What do you want me to talk about in
the review?
Experiment 3
• Remember you have to prepare in your
notebook the volumes you are going to
use for your restriction digestion.
Assignment 4
•
PART 1: Experiment 3
•
Page 1 Will present 3 labeled gels: 1 the gel of the digest of the vector and
digest for DNA fragment isolation; 2 the gel of the ligation reactions and isolated
DNA fragment; 3 the gel of the restriction digestions of the two clones.
•
Page 2 You will summarize the success or failure of each step of the process of
cloning.
•
PART 2: Experiment 5
•
Page 3-4 will be your report on the yeast mutant screen. The text following the
table shall answer four questions about the mutants you characterized.
Summary of Expt. 5 in your notebook
1
2
3
4
5
6
7
8
P
Initial Phenotype
of Mutant
Complementation
Test
beta-galactosidase
Test
Probable Mutation
Red or White
Red or White
Blue or White
swi5, swi, she or
unknown
Notebook
• Look at the instructions and expectations on
the web site.
• Goal is to have a document that you can
show a potential employer or supervisor
demonstrating what you have experience
doing and that you have the ability to record
data in a true functioning lab notebook.
The LacZ Reporter Gene
beta-galactosidase is an enzyme encoded by the bacterial gene
lacZ.
beta-galactosidase cleaves the colorless substrate X-gal (5bromo-4-chloro-3-indolyl-galactopyranoside) into galactose
and a blue insoluble product of the cleavage.
Therefore, lacZ can be used as a reporter gene (blue/white
selection)
X-GAL
Cis-acting elements
URS 1
URS 2
lacZ
GAL1-10
URS 2
lacZ
Cis-acting elements
lacZ
Mother daughter
URS 2
lacZ
Late G1 in both
mothers and
daughters
GAL1-10
lacZ
URS 1
Galactose inducible
in both mothers and
daughters.
Apart the elements are independent; together the elements are
restrictive and not additive.
Properties
URS 1
URS 2
lacZ
*Haploid specific
*Cell cycle regulated
Mother cell specific
GAL1-10
URS 2
*Haploid specific
*Cell cycle regulated
Galactose inducible
lacZ
Trans-acting factors required for expression
Cis element
URS1
Trans acting factors
Swi5p
Swi1p, Swi2p, Swi3p
URS2
Swi4p, Swi6p
Swi1p, Swi2p, Swi3p
GAL1-10
Gal4p
Therefore in swi5lf or shelf mutant haploid cells grown on
Galactose plates:
URS 1
URS 2
CAN1
Is not expressed
GAL1-10
URS 2
lacZ
Is expressed
BLUE
Therefore, in the other swi mutant haploids grown on
Galactose plates
URS 1
URS 2
CAN1
Is not expressed
GAL1-10
URS 2
lacZ
Is not expressed
WHITE
Summary of Expt. 5 in your notebook
1
2
3
4
5
6
7
8
P
Initial Phenotype
of Mutant
Complementation
Test
beta-galactosidase
Test
Probable Mutation
Red or White
Red or White
Blue or White
swi5, swi, she or
unknown
What would you like me to explain to you now, and
what would you like me to review next lecture?