T9: Molecular Characterization of an T9: Molecular Characterization of an Unknown P-element Insertion in Unknown P-element Insertion in

Drosophila melanogasterDrosophila melanogaster

Finding a Gene’s FunctionFinding a Gene’s Function

Easy to sequence genesEasy to sequence genes– Hard to figure out their functionHard to figure out their function

How to find a gene’s functionHow to find a gene’s function– Mutate the geneMutate the gene– Identify the defect caused by the mutationIdentify the defect caused by the mutation– Identify which gene was mutatedIdentify which gene was mutated

Insertional MutagenesisInsertional Mutagenesis– Uses transposons to interrupt a gene Uses transposons to interrupt a gene

sequence, making it function improperlysequence, 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} TransposonThe P{lacW} Transposon

Mutates by insertional mutagenesisMutates by insertional mutagenesis– Adjacent to mutated geneAdjacent to mutated gene

Gives flies red eye for easy identificationGives flies red eye for easy identification Contains a plasmid, allowing us to clone the geneContains a plasmid, allowing us to clone the gene Restriction enzyme sites strategically placedRestriction enzyme sites strategically placed

The pBR322 plasmidThe pBR322 plasmid

Origin of ReplicationOrigin of Replication– Allows for replication to Allows for replication to

occuroccur Taken up by Taken up by E. coliE. coli Ampicillin ResistanceAmpicillin Resistance

– Allows only bacteria Allows only bacteria with gene to livewith 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 EcoRIEcoRI

P element 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

(6) Purify & analyze cloned genomic DNA

E. coli bacterium

Restriction enzymesRestriction enzymes

cut double-stranded DNAcut double-stranded DNA cut only at a specific nucleotide sequence or cut only at a specific nucleotide sequence or recognition sequencerecognition sequence sequences typically four to twelve nucleotides sequences typically four to twelve nucleotides longlong

EcoRI restriction enzyme cut sequence

P{lacW} transposon

LigationLigation

P{lacW} (ligase added)

TransformationTransformation ““genetic alteration of a genetic alteration of a

cell resulting from the cell resulting from the introduction, uptake and introduction, uptake and expression of foreign expression of foreign DNA” **DNA” **– If Bacteria pick up If Bacteria pick up

pBR322 pBR322 ampampRR

– if not if not can’t grow on can’t grow on ampicillinampicillin

Escherichia coliEscherichia coli– Grows really quicklyGrows really quickly– Can be made Can be made

competentcompetent Competence Competence

– Ability to uptake DNA Ability to uptake DNA from the environmentfrom the environment

**http://encyclopedia.laborlawtalk.com/Transformation

A.A. PreparationPreparation1)1) Treatment with RuClTreatment with RuCl2 2

2)2) Heat shockHeat shock3)3) Ethanol precipitation & centrifugationEthanol precipitation & centrifugation4)4) Transformation – DH5 alpha strainTransformation – DH5 alpha strain

B.B. PlatingPlating1)1) Streak onto L-amp50 plateStreak onto L-amp50 plate

Transformation - methodTransformation - method

What HappenedWhat Happened

ControlsControls E. coli,E. coli, no plasmid no plasmid

– No bacterial growthNo bacterial growth

Digestion and Ligation, no plasmidDigestion and Ligation, no plasmid– No bacterial growthNo bacterial growth

Ampicillin is effectiveE. Coli is not naturally ampicillin resistantNo DNA contamination of digestion and ligation reagents

Controls (cont.)Controls (cont.) Plasmid and Plasmid and E. coliE. coli

– Bacterial lawnBacterial lawn Digestion and Ligation, plasmidDigestion and Ligation, plasmid

– Bacterial coloniesBacterial colonies Digestion, ligation (no ligase), plasmidDigestion, ligation (no ligase), plasmid

– No growthNo growth

Bacterial Lawn

Bacterial Colonies

E. Coli is competent----------

Transformation process works

Either Digestion & Ligation workedOR

Neither workedDigestion worked (?)

Isolation of gDNA worked---------

Ligation and/or digestion failed

What we would have doneWhat we would have done

Isolation of plasmid (w/ gDNA)Isolation of plasmid (w/ gDNA) Restriction MappingRestriction Mapping

– Gel ElectrophoresisGel Electrophoresis– Figure out length of gDNA segmentFigure out length of gDNA segment

Sequencing of gDNA segmentSequencing of gDNA segment BioinformaticsBioinformatics

What we learnedWhat we learned How to work with How to work with DrosophilaDrosophila Microbiology Microbiology

– TransposonsTransposons– MutationsMutations– Plasmid use and isolationPlasmid use and isolation– TransformationTransformation

Proper lab techniquesProper lab techniques– Micropipetting Micropipetting – Gel electrophoresisGel electrophoresis– Plating bacteriaPlating bacteria– Sterile technique, reagent handlingSterile technique, reagent handling

In biological research, success is not guaranteed.In biological research, success is not guaranteed.

BEWARE OF FORREST FIRESBEWARE OF FORREST FIRES

Thank You!Thank You!

Dr. CookDr. Cook KirstenKirsten Dr. MiyamotoDr. Miyamoto Paul H. and Dr. SuracePaul H. and Dr. Surace Nerds like youNerds like you