mutation of prx 1 on thioredoxin 90 ashley morris
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- MUTATION OF PRX 1 ON THIOREDOXIN 90 Ashley Morris
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- Peroxiredoxins 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.
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- Peroxiredoxins continued Peroxiredoxins (Prx) are present in organisms from all kingdoms located on a terminal that is the primary site of oxidation by H 2 O 2
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- T90 This protein has now been shown to be phosphorylated specifically on T90 by several CDKs including Cdc2, incitro. Phosphorylation of Prx1 on T90 reduced the peroxidase activity of protein by 80%.
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- Purpose Was to mutate the gene of interest
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- Method Primers how/why they were designed Whats going on in the thermal cycler Amplify the DNA with mutated gene Primes Replication of new gene Digestion Transformation Growth on plates
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- 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
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- 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.
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- Results On agar plate T90D 63 colonies and T90A and no colonies 63 colonies No growth
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- 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 1195 C30 seconds 21895 C 68 C 30 seconds 1 minute 4 minutes 20 seconds 3168 C 4 C 15 minutes
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- 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.
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- Results No Growth We wrapped our plates and placed them in the incubator, which youre not supposed to do because the bacteria cannot breathe so it cant go.
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- No Growth
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- GEL We ran our DNA control and DNA sample and our mutated samples on a gel Quantification
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- Results LanesSamples 1Control 2T90A #2 3T90D #2 4N/A 5 6DNA 7T90A #1 8T90D #1 9N/A 10N/A
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- 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.
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- Results I had no growth on any of my plates
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- 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.
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- 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.
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- 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:
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- Sources http://www.rndsystems.com/pdf/AF3488.pd http://www.rndsystems.com/pdf/AF3488.pd http://www.nwlifescience.com/index.php?page=sho p.product_details&flypage=flypage.tpl&product_i d=41&category_id=9&option=com_virtuemart&Ite mid=256f http://www.nwlifescience.com/index.php?page=sho p.product_details&flypage=flypage.tpl&product_i d=41&category_id=9&option=com_virtuemart&Ite mid=256f http://en.wikipedia.org/wiki/PRDX4 http://en.wikipedia.org/wiki/PRDX4
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