Molecular Characterization of a P-element Insertion in Drosophila melanogaster: Insights and Techniques

1. 0 T9: Molecular Characterization of an Unknown P-element Insertion in Drosophila melanogaster

2. 0 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

3. 0 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

4. 0 The P{lacW} Transposon • 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

5. 0 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

6. 0 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

7. 0 EcoRI EcoRI EcoRI EcoRI 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

8. 0 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

9. 0 Ligation P{lacW} (ligase added)

10. 0 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.laborlawtalk.com/Transformation

11. 0 Transformation - method • Preparation • Treatment with RuCl2 • Heat shock • Ethanol precipitation & centrifugation • Transformation – DH5 alpha strain • Plating • Streak onto L-amp50 plate

12. 0 What Happened ?

13. Controls • E. coli, no plasmid • No bacterial growth • Digestion and Ligation, no plasmid • No bacterial growth Ampicillin is effective E. Coli is not naturally ampicillin resistant No DNA contamination of digestion and ligation reagents

14. Bacterial Colonies 0 Controls (cont.) Bacterial Lawn • Plasmid and E. coli • Bacterial lawn • Digestion and Ligation, plasmid • Bacterial colonies • Digestion, ligation (no ligase), plasmid • No growth E. Coli is competent ---------- Transformation process works Either Digestion & Ligation worked OR Neither worked Digestion worked (?) Isolation of gDNA worked --------- Ligation and/or digestion failed

15. 0 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

16. 0 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.

17. 0 BEWARE OF FORREST FIRES

18. 0 Thank You! • Dr. Cook • Kirsten • Dr. Miyamoto • Paul H. and Dr. Surace • Nerds like you