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 H2O2

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 colonies63

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.Segme

nt Cycles Temperature Time

1 1 95º C 30 seconds

2 18 95º C68º C68º C

30 seconds1 minute4 minutes 20 seconds

3 1 68º C4º 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

Lanes

Samples

1 Control

2 T90A #2

3 T90D #2

4 N/A

5 N/A

6 DNA

7 T90A #1

8 T90D #1

9 N/A

10 N/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

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