Download Drosophila Melanogaster

T9: Molecular Characterization of an
Unknown P-element Insertion in
Drosophila melanogaster
Finding a Gene’s Function
 Easy
to sequence genes
– Hard to figure out their function
 How
to find a gene’s function
– Mutate the gene
– Identify the defect caused by the mutation
– Identify which gene was mutated
 Insertional
Mutagenesis
– Uses transposons to interrupt a gene
sequence, making it function improperly
Drosophila Melanogaster
 Model
organism for over 90 years
– Commonly known as the fruit fly
– Complete genome sequenced in 2000
– 4 pairs of chromosomes and about
14,000 genes
– Matures quickly
– Reproduces rapidly
– Large number of progeny
The P{lacW} Transposon



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Mutates by insertional mutagenesis
– Adjacent to mutated gene
Gives flies red eye for easy identification
Contains a plasmid, allowing us to clone the
gene
Restriction enzyme sites strategically placed
The pBR322 plasmid



Origin of Replication
– Allows for
replication to
occur
Taken up by E. coli
Ampicillin Resistance
– Allows only
bacteria with gene
to live
P Element Plasmid Rescue
 Our
goal using Plasmid Rescue is to:
– Recover and identify the insertion site of
a P-element in the fruit fly genome
 There
are four basic steps:
– Isolation of genomic DNA
– Cleavage of genomic DNA with
restriction enzymes
– Ligation of cleaved DNA
– Uptake of ligated DNA by E. coli
EcoRI
EcoRI
P element
EcoRI
EcoRI
Gene segment
(1) Purify genomic DNA
(2) Digest a restricton enzyme, e.g. EcoRI
(3) Ligate the digested DNA
(4) Transform into Escherichia coli bacteria
(5) Select drug
resistant bacteria
E. coli bacterium
(6) Purify & analyze
cloned genomic
DNA
Restriction enzymes
P{lacW} transposon
cut double-stranded DNA
 cut only at a specific nucleotide sequence or
recognition sequence
 sequences typically four to twelve nucleotides
long

EcoRI restriction enzyme cut sequence
Ligation
P{lacW} (ligase added)
Transformation



“genetic alteration of a
cell resulting from the
introduction, uptake and
expression of foreign
DNA” **
– If Bacteria pick up
pBR322 ampR
– if not  can’t grow on
ampicillin
Escherichia coli
– Grows really quickly
– Can be made
competent
Competence
– Ability to uptake DNA
from the environment
**http://encyclopedia.laborlaw
talk.com/Transformation
Transformation - method
A.
Preparation
1)
2)
3)
4)
B.
Treatment with RuCl2
Heat shock
Ethanol precipitation & centrifugation
Transformation – DH5 alpha strain
Plating
1) Streak onto L-amp50 plate
What Happened
Controls
 E.
coli, no plasmid
–No bacterial growth
 Digestion
and Ligation, no plasmid
–No bacterial growth
Ampicillin
is effective of digestion and
No DNA contamination
ligation
E. Coli isreagents
not naturally ampicillin resistant
Bacterial
Lawn
Controls (cont.)
Plasmid and E. coli
– Bacterial lawn
 Digestion and Ligation, plasmid
– Bacterial colonies
 Digestion, ligation (no ligase), plasmid
– No growth

Either
Digestion
& Ligation
worked
Isolation
E.
Coli of
is gDNA
competent
worked
OR
Digestion
-----------------worked (?)
Neither
worked
Ligation
Transformation
and/or digestion
process works
failed
Bacterial
Colonies
What we would have done
 Isolation
of plasmid (w/ gDNA)
 Restriction Mapping
–Gel Electrophoresis
–Figure out length of gDNA segment
 Sequencing of gDNA segment
 Bioinformatics
What we learned




How to work with Drosophila
Microbiology
– Transposons
– Mutations
– Plasmid use and isolation
– Transformation
Proper lab techniques
– Micropipetting
– Gel electrophoresis
– Plating bacteria
– Sterile technique, reagent handling
In biological research, success is not guaranteed.
BEWARE OF FORREST FIRES
Thank You!
Dr. Cook
 Kirsten
 Dr. Miyamoto
 Paul H. and Dr. Surace
 Nerds like you
