MUTATION OF PRX 1 ON THIOREDOXIN 90 Ashley Morris

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.

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 H 2 O 2

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%.

Purpose  Was to mutate the gene of interest

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

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 used instead of the recommended XL1- Blue supercompetent cells

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.

Results  On agar plate T90D 63 colonies and T90A and no colonies 63 colonies No growth

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. SegmentCyclesTemperatureTime 1195º C30 seconds 21895º C 68º C 30 seconds 1 minute 4 minutes 20 seconds 3168º C 4º C 15 minutes ∞

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.

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.

No Growth

GEL  We ran our DNA control and DNA sample and our mutated samples on a gel  Quantification

Results LanesSamples 1Control 2T90A #2 3T90D #2 4N/A 5 6DNA 7T90A #1 8T90D #1 9N/A 10N/A

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.

Results  I had no growth on any of my plates

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.

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.

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:

Sources   p.product_details&flypage=flypage.tpl&product_i d=41&category_id=9&option=com_virtuemart&Ite mid=256f p.product_details&flypage=flypage.tpl&product_i d=41&category_id=9&option=com_virtuemart&Ite mid=256f 