CPD-Y - Senior Seminar (2)

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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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