Mutation of Prx 1 on Thioredoxin 90

1. Mutation of Prx 1 on Thioredoxin 90 Ashley Morris

2. Peroxiredoxin’s 1Information • Peroxiredoxins: • family of antioxidant proteins sharing a common reactive Cys residue in the N-terminal region • capable of serving as a peroxidase • Peroxiredixin 1 is the isoform found in the cytosol of cells. • Peroxidases of the peroxiredoxin family reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohol. • The protein is localized to the cytoplasm.

3. Peroxiredoxin’s continued • Peroxiredoxin’s (Prx) are present in organisms from all kingdoms • located on a terminal that is the primary site of oxidation by H2O2

4. T90 • This protein has now been shown to be phosphorylated specifically on T90 by several CDK’s including Cdc2, incitro. • Phosphorylation of Prx1 on T90 reduced the peroxidase activity of protein by 80%.

5. Purpose • Was to mutate the gene of interest

6. Method • Primers – how/why they were designed • What’s going on in the thermal cycler • Amplify the DNA with mutated gene • Primes • Replication of new gene • Digestion • Transformation • Growth on plates

7. Prx 1 Trial One • We calculated the melting temperature of our primers by using the formula given in the QuickChange II packet • We followed the procedure from QuickChange II Site Directed Mutagenesis Kit with a few exceptions: • 5X reaction buffer was used instead of 10x • NEB 5-alpha competent E. Coli was usedinstead of the recommended XL1- Blue supercompetent cells

8. Trial One Continued • The PRC machine that held our samples was incorrectly set • We used High Efficiency Transformation Protocol instead of the suggested protocol in the packet. • We then added Dpn 1 to the tubes to digest the reaction • Transformation • Soon after we transferred our cells onto agar plates and placed them at 37 degrees and allowed them to grow.

9. Results • On agar plate T90D 63 colonies and T90A and no colonies No growth 63 colonies 63 colonies

11. Trial 2 • We followed the suggested procedure from QuickChange II Site Directed Mutagenesis Kit including the control plasmid. • The PCR temperatures and times were set by the following guide line.

12. Trial Two Continued • One additional step we had was to make NZY+ broth for our bacteria to be cultivated in. • After the amplification we transformed our mutated cells and the control into XL1- Blue supercompetent cells on agar plates that had ampicillin.

13. Results • No Growth • We wrapped our plates and placed them in the incubator, which you’re not supposed to do because the bacteria cannot breathe so it can’t go.

14. No Growth

15. GEL • We ran our DNA control and DNA sample and our mutated samples on a gel • Quantification

16. Results

17. Trial 3 • I used the procedure from the QuickChange II Site Directed Mutagenesis Kit. • I cut all of the components needed for each reaction in half.

18. Results • I had no growth on any of my plates

19. Further Research • We would need to repeat the experiment multiple times to see if we could get consistent growth on the plates. • We would need to try both types of cells because for my experiment I only had growth on the NEB 5-alpha competent E. Coli cells and not the XL1- Blue supercompetent. That could be a factor in this project.

20. Further Research Continued • Our NZY+ broth could have been incorrect. • We need to test both of the procedures we used and set the experiments up exactly the same and run them at the same time.

21. Expected Results • This experiment was difficult • I had plates that grew so we will have to send them off for sequencing. • If this had worked, the next step is:

22. Sources • http://www.rndsystems.com/pdf/AF3488.pd • http://www.nwlifescience.com/index.php?page=shop.product_details&flypage=flypage.tpl&product_id=41&category_id=9&option=com_virtuemart&Itemid=256f • http://en.wikipedia.org/wiki/PRDX4