cpd-y - senior seminar (2)
TRANSCRIPT
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Exploring Cutting Edge Biotechnology:
A Cloning Blueprint of Serine Carboxypeptidase Y
Research Advisor: Dr. Mary A. Kopecki-Fjetland
Researcher: Perouza Parsamian
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Overview• Background
• Research Goal• Prior Research
• Methodology + Results• Conclusion• Future work
• Acknowledgements
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Background
Serine Carboxypeptidase Y (CPD-Y)
CN
Multifunctional• Transpeptidation• Gen. turnover• Free amino acids• Intracellular enzymes
Serine-257 Aspartate-449 Histidine-508
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Background
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Background cont.
Serine Protease (Trypsin)
CN
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Research GoalConvergent Evolution ?
CN CNEndopeptidase – Serine Protease Exopeptidase – CPD-Y
Immediate goal:Confirm the CPD-Y gene is present in the construct.a) Grow cellsb) Isolate construct (DNA mini prep)c) Cut CPD-Y gene out of constructd) Visualize digested DNA and quantize
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Prior Research
AAAAAA
TTT
TTT
Saccharomyces cerevisiae
CPD-Y Gene
pGEM-T vector
CPD-Y + pGEM-T
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Prior Research
EcoR1EcoR1
CPD-Y Gene
CPD-Y + pGEM-T
CPD-Y + pGEM-Tw/ EcoR1 restriction
Enzyme
pET-32c Vector
CPD-Y + pET-32c
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EcoR1EcoR1
Current ResearchMethodology – 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
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Current ResearchMethodology – A) Grow cells
CPD-Y + pET-32c construct in E.coli
LB Broth
5mL LB Broth +5 µL of
Ampicillin(50microg/ml)
Incubate/shake at 37°C
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Current ResearchResults – 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
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Current ResearchResults – A) Grow cells - Transformation
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Current ResearchResults – A) Grow cells - Transformation
Date Plate #1 Plate #2 Plate #3
6/22/12 Test plasmid(+)
Ratio #1:3 (+) Ratio#1:7 (+)
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Current ResearchResults – A) Grow cells - Transformation
Date TT#1 TT#2 TT#3 TT#4 TT#5 TT#6 TT#7 TT#8 TT#96/25/12 (+) (+) (+) (+) (+) (+) (+) pUC Neg
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Current ResearchMethodology – A) Mini-Prep Procedure - Purification
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.
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Current ResearchMethodology – A) Mini-Prep Procedure - Purification
6. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column toempty by gravity flow.7. Apply the supernatant from step 4 to the QIAGEN-tip 20 and allow it to enter theresin by gravity flow.8. Wash the QIAGEN-tip 20 with 2 x 2 ml Buffer QC9. Elute DNA with 0.8 ml Buffer QF.10. Precipitate DNA by adding 0.56 ml ofroom-temperature isopropanol to the eluted DNA. Mix and centrifuge immediatelyfor 30 min in a micro-centrifuge. Carefully decant the supernatant.
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Current ResearchMethodology – A) Mini-Prep Procedure - Purification
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)
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Current ResearchMethodology – B) Digest construct
EcoR1EcoR1
CPD-Y + pET-32c
1) CPD-Y was digested using DI water2) 10X EcoR1 Buffer3) DNA Construct4) EcoR1 restriction Enzyme at the EcoR1 site5) Incubated overnight at (37ºC)
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Current ResearchMethodology – C) Visualize DNA and Quantify
CPD-Y Gene
pET-32c Vector
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Current ResearchMethodology – C) Visualize DNA and Quantify
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Current ResearchResults – D) Visualize DNA & Quantify
Agilent Bioanalyzer- Electropharagram
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Current ResearchResults – E) 6 Weeks of findings
Agilent Bioanalyzer- pUC18-June 11th sample
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Current ResearchResults – D) Visualize DNA & Quantify
Gel Electrophoresis
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Current ResearchResults – D) Visualize DNA & Quantify
Gel electrophoresis - pUC18-June 11th sample
3000 Kb
Perouza Dr. K
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ConclusionPositive 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.
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Future work• Confirmation of Clones• Sequence DNA• Kinetic Studies
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Acknowledgements
Dr. Mary Kopecki-Fjetland Katharina Weber
Everyone!
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