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

2. 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. 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. 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. 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. 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. EcoRI P elementGene 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. Restriction enzymes  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 P{lacW} transposon

9. Ligation P{lacW} (ligase added)

10. Transformation  “genetic alteration of a cell resulting from the introduction, uptake and expression of foreign DNA” ** –If Bacteria pick up pBR322  amp R –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

11. A. Preparation 1)Treatment with RuCl 2 2)Heat shock 3)Ethanol precipitation & centrifugation 4)Transformation – DH5 alpha strain B. Plating 1)Streak onto L-amp50 plate Transformation - method

12. 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. Controls (cont.)  Plasmid and E. coli –Bacterial lawn  Digestion and Ligation, plasmid –Bacterial colonies  Digestion, ligation (no ligase), plasmid –No growth Bacterial Lawn Bacterial Colonies 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. 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. 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. BEWARE OF FORREST FIRES

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