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
