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CPD-Y - Senior Seminar (2)
- 1. Exploring Cutting Edge Biotechnology: A Cloning Blueprint of Serine Carboxypeptidase Y 1 Research Advisor: Dr. Mary A. Kopecki-Fjetland Researcher: Perouza Parsamian
- 2. Overview • Background • Research Goal • Prior Research • Methodology + Results • Conclusion • Future work • Acknowledgements 2
- 3. Background 3Serine Carboxypeptidase Y (CPD-Y) CN Multifunctional • Transpeptidation • Gen. turnover • Free amino acids • Intracellular enzymes Serine-257 Aspartate-449 Histidine-508
- 4. Background 4
- 5. Background cont. 5Serine Protease (Trypsin) CN
- 6. Research Goal 6 Convergent Evolution ? CN CN Endopeptidase – Serine Protease Exopeptidase – CPD-Y Immediate goal: Confirm the CPD-Y gene is present in the construct. a) Grow cells b) Isolate construct (DNA mini prep) c) Cut CPD-Y gene out of construct d) Visualize digested DNA and quantize
- 7. Prior Research 7 AAA AAA TTT TTT Saccharomyces cerevisiae CPD-Y Gene pGEM-T vector CPD-Y + pGEM-T
- 8. Prior Research 8 EcoR1EcoR1 CPD-Y Gene CPD-Y + pGEM-T CPD-Y + pGEM-T w/ EcoR1 restriction Enzyme pET-32c Vector CPD-Y + pET-32c
- 9. 9 EcoR1EcoR1 Current Research Methodology – Confirm CPD-Y is present in the construct CPD-Y + pET-32c CPD-Y + pET-32c w/ EcoR1 restriction enzyme CPD-Y Gene pET-32c Vector Analyze
- 10. Current Research Methodology – A) Grow cells 10 CPD-Y + pET-32c construct in E.coli LB Broth 5mL LB Broth + 5 µL of Ampicillin(50microg/ml) Incubate/shake at 37°C
- 11. 11 Current Research Results – A) Grow cells Date W#1 W#2 W#3 W#4 W#5 W#6 W#7 W#8 W#9 W#10 5/24/12 (-) (-) (-) (-) (-) (-) (+) (-) N/A N/A 5/29/12 N/A N/A N/A N/A N/A N/A (-) N/A N/A N/A 5/31/12 N/A N/A N/A N/A N/A N/A (30μL) (-) N/A N/A N/A 6/11/12 (50μL) (-) (50μL) (-) (50μL) (-) (50μL) (-) (50μL) (-) (50μL) (-) (50μL) (-) (50μL) (-) N/A N/A 6/14/12 500μL (-) 500μL (-) (500μL (-) 500μL (-) N/A N/A 200μL (-) N/A 100μL (-) 100μL (-) 6/18/12 (All) (-) (All) (+) (All) (-) (All) (-) 500μL (-) 500μL (-) (All) (-) (500μL ) (-) (All) (-) (All) (-) 6/19/12 N/A (-) N/A N/A (All) (-) (All) (-) N/A (All) (-) N/A N/A
- 12. 12 Current Research Results – A) Grow cells - Transformation
- 13. 13 Current Research Results – A) Grow cells - Transformation Date Plate #1 Plate #2 Plate #3 6/22/12 Test plasmid (+) Ratio #1:3 (+) Ratio #1:7 (+)
- 14. 14 Current Research Results – A) Grow cells - Transformation Date TT#1 TT#2 TT#3 TT#4 TT#5 TT#6 TT#7 TT#8 TT#9 6/25/12 (+) (+) (+) (+) (+) (+) (+) pUC Neg
- 15. Current Research Methodology – A) Mini-Prep Procedure - Purification 15 1. Harvest the bacterial cells by centrifugation for 15 min. 2. Re-suspend the bacterial pellet in 0.3 ml of Buffer P1. 3. Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times, and incubate at room temperature (15–25°C) for 5 min. 4. Add 0.3 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 5 min. 5. Centrifuge at maximum speed in a micro-centrifuge for 10 min. Remove supernatant containing plasmid DNA promptly.
- 16. Current Research Methodology – A) Mini-Prep Procedure - Purification 16 6. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column to empty by gravity flow. 7. Apply the supernatant from step 4 to the QIAGEN-tip 20 and allow it to enter the resin by gravity flow. 8. Wash the QIAGEN-tip 20 with 2 x 2 ml Buffer QC 9. Elute DNA with 0.8 ml Buffer QF. 10. Precipitate DNA by adding 0.56 ml of room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately for 30 min in a micro-centrifuge. Carefully decant the supernatant.
- 17. Current Research Methodology – A) Mini-Prep Procedure - Purification 17 11. Wash DNA pellet with 1 ml of 70% ethanol and centrifuge for 10 min. Carefully decant the supernatant without disturbing the pellet. 12. Air-dry the pellet redissolve the DNA in a suitable volume of buffer (10mM Tris·Cl, pH 8.5)
- 18. Current Research Methodology – B) Digest construct 18 EcoR1EcoR1 CPD-Y + pET-32c 1) CPD-Y was digested using DI water 2) 10X EcoR1 Buffer 3) DNA Construct 4) EcoR1 restriction Enzyme at the EcoR1 site 5) Incubated overnight at (37ºC)
- 19. Current Research Methodology – C) Visualize DNA and Quantify 19 CPD-Y Gene pET-32c Vector
- 20. Current Research Methodology – C) Visualize DNA and Quantify 20
- 21. 21 Current Research Results – D) Visualize DNA & Quantify Agilent Bioanalyzer- Electropharagram
- 22. 22 Current Research Results – E) 6 Weeks of findings Agilent Bioanalyzer- pUC18-June 11th sample
- 23. 23 Current Research Results – D) Visualize DNA & Quantify Gel Electrophoresis
- 24. 24 Current Research Results – D) Visualize DNA & Quantify Gel electrophoresis - pUC18-June 11th sample 3000 Kb Perouza Dr. K
- 25. 25 Conclusion Positive improvements + Achievements • Improve mini-prep technique - Check • Improve Agilent bioanalyzer technique – improvement • Improve Gel Electrophoresis technique – Check • Achieved one peak results – Agilent bioanalyzer • Achieved clearer DNA bands – Gel Electrophoresis • Continually improving problem solving skills and watching for patterns • Achieved a new outlook on research – results are just a bonus, after techniques are mastered through constant repetition.
- 26. 26 Future work • Confirmation of Clones • Sequence DNA • Kinetic Studies
- 27. 27 Acknowledgements Dr. Mary Kopecki-Fjetland Katharina Weber Everyone!
- 28. 28
Editor's Notes
- Good afternoon
- Today’s presentation contains a brief review of the…
- Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. There are two groups of Carboxypeptidases, metallo and serine carboxypeptidases. Serine carboxypeptidase is our primary enzyme of discussion, more specifically CPD-Y. CPD-Y is found in the vacuoles of Saccharomyces cerevisiae, commonly known as bakers yeast. Not only do they function as exopeptidases but they also participate in transpeptidation reactions, general turnover of proteins, providing free amino acids from storage proteins and play a role as intracellular enzymes. CPD-Y has a catalytic triad consisting of Serine-257, Aspartate-449, and Histidine-508, respectively. (talk about positions)
- Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. There are two groups of Carboxypeptidases, metallo and serine carboxypeptidases. Serine carboxypeptidase is our primary enzyme of discussion, more specifically CPD-Y. CPD-Y is found in the vacuoles of Saccharomyces cerevisiae, commonly known as bakers yeast. Not only do they function as exopeptidases but they also participate in transpeptidation reactions, general turnover of proteins, providing free amino acids from storage proteins and play a role as intracellular enzymes. CPD-Y has a catalytic triad consisting of Serine-257, Aspartate-449, and Histidine-508, respectively. (talk about positions)
- A similar enzyme Serine Protease, also known as Serine Endopeptidase, is similar in structure to CPD-Y, although functions differently by cleaving in between the polypeptide chain rather than the C-Terminal end.
- This presents a phenomenon known as Convergent evolution where there are similarities in function however variance in structure. The immediate goal is to confirm that the CPD-Y gene is present within the construct. To accomplish this goal, we have to grow our E.coli cells, isolate our construct via DNA mini-prep, cut the CPD-Y gene out of the construct and finally visualize digested DNA and quantize through different analytical methods. Another analytical technique is to design a Polymerase Chain Reaction (PCR) experiment to visualize the presence of our gene.
- From prior research, CPD-Y was extracted from Saccharomyces cerevisiae and ligated into a non-expression vector, pGEM-T, This non-expression vector was utilized to provide overhangs complementary to PCR product. In addition, this vector yields a high copy number and prevents unintended expression of the protein.
- Once confirmed that CPD-Y was present in the non-expression construct, the gene was re-isolated using EcoR1 restriction enzyme, purified and ligated into an expression vector, pET-32C at the EcoR1 site.
- Our next step is to confirm CPD-Y is present in the construct, through different analytical techniques, such as using the Agilent bioanalyzer.
- To grow E.coli cells with our construct we used a nutritious media, LB Broth. We aquiloted 5ml of LB broth and 5 microL of ampicillin into sterilized test tubes and inocuated our cells and placed them into the incubator/shaker overnight at 37 degrees Celsius an optimal temperature for growing ecoli cells. The use of ampicillin was to insure no other bacteria would grow as our vector had ampicillin resistance.
- As you can see from this table most if not all our tests were unsuccessful. The positive outcomes that we did achieve were inconsistent in growth as the glycerol stocks in which we recovered our cells were improperly made. As the weeks went by we increased the amount of volume we inoculated into our media. Our results continued to show no growth. For the first week we placed a lot of emphasis on Whitney’s glycerol stock #7, It was promising at first then no longer showed any progress as we increased the volume of cells. This was also the same outcome in regards to Glycerol stock #2 found on June 18th.
- The next plan of action was to do a fresh transformation of our construct. We took ratios of 1:7, 1:3, a test plasmid and transformed them into and compentant E.coli cell. By adding 1micoL of our construct into competent Ecoli cells and adding 250 micoL of SOC medium, which is another nutritious medium for competent cells, therefore creating our transformated cells. We then streaked our transformed construct onto an LB agar and ampicillin plate. Added a “pool” of SOC medium and 25 micoL of our transformed construct and Incubated at 37ºC overnight.
- Our results showed sufficient growth on the plates. The numbers are the single colonies I had selected to grow in liquid culture. Each plate is labeled with its designated test. (left) that was the test plasmid, (middle) 1:3 ratio and (right) 1:7 ratio. Only one colony had grown for the 1:3 plate. A result of a low colony count indicates a possibility that the CDP-Y gene had been in the freezer for a long time and the death of the gene occurred.
- Inoculating our cells into liquid cultures overnight and achieving growth proves the transformation protocol was conducted correctly.
- CPD-Y was digested using DI water 10X EcoR1 Buffer DNA Construct EcoR1 restriction Enzyme at the EcoR1 site Incubated overnight at (37ºC)
- Once our gene is digested, isolated and linearized. The DNA was visualized and quantized by the agilent bioanalyzer and gel electrophoresis.
- An Electropharagam of a 1 Kb ladder that was tested. As you can see the electropharagram is measured by florescence on the y-axis and seconds on the x-axis. Each peak has a Base pair number, which is the number of nitrogenous bases in each fragment. The intensity of the peak is based on the concentration of the fragment. And the higher the concentration the higher in florecence of the conjugated nitrogenous bases. And it goes from smallest basepair size to largest basepair size.
- Another visualization and Quantization technique is to use Gel Electrophoresis. DNA samples are bound to dyes, driven by a voltage and separated by size in a prepared 1% agarose gel. A UV camera is used to visualize the resulting bands.
- I would like to thank Dr. Kopecki, for giving me this opportunity and being an inspirational mentor to me throughout this research, Katharina weber for being a fantastic student and research partner of whom worked with me last summer, the chemistry Welch foundation for funding this project and Everyone who is on this journey with me! Thank-you for listening!