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Plasmid Construction and Purification of Arabidopsis Plantacyanin and Lily
Chemocyanin in a Prokaryotic System
Suhyen Lee and Brooke Vuong (Aram Nersissian)
Outline
•Purpose•Arabidopsis Plantacyanin•Lily Chemocyanin•Chemotropism Assay•pET-3a vector•Mechanism-Plasmid Construction
-PCR Condition -DNA purification after PCR -DNA digestion and ligation -Transformation -Protein Purification
Purpose
collaborate with UCR to purify protein so that UCR can use purified protein to do further research on Arabidopsis Plantacyanin and Lily Chemocyanin
• A basic, 10kD, copper binding …..protein with pI ~10• Characterized by turning blue upon …..binding copper in its +2 oxidation …..state • Its function remains unknown• Has a single copper binding site …..formed by four ligands: two …..histidines and a cystein …..coordinate equatorially, while the …..fourth, axial ligand, is a …..methionine or a glutamine.• The redox potential of plantacyanin …..bound copper varies with the …..hydrophobicity of the axial ligand: …..it increases along with the …..hydrophobicity of the ligand
Arabidopsis Plantacyanin
http://aggie-horticulture.tamu.edu/faculty/davies/students/ngo/research.html
http://www.pnas.org/misc/3800.jpg
• A member of the plantacyanin family ….copper binding proteins • Implicated in Chemotropism in lily ….stigma• Essential in pollination: pollen tube ….chemotropism induction• Shows 67% sequence similarity to ….plantacyanin• Copper binding site: two histidines and ….a cysteine. In place of the axial ligand ….has a non-coordinating, hydrophobic ….leucine residue. • Copper binding and other biochemical ….and biophysical properties have not ….been characterized.
Lily Chemocyanin
Copyright ©2003 by the National Academy of Sciences
Kim, Sunran et al. (2003) Proc. Natl. Acad. Sci. USA 100, 16125-16130
Fig. 3. Chemotropism assay showing pollen tube reorientation over time (A and B) and quantification method (C)
pET-3a Vector
*Designed for cloning and recombinant protein expressions in prokaryotic system, E. Coli.*Utilizes T7 RNA polymerase in the host cell to transcribe and translate. *Has NdeI and BamHI restriction sites which make directional cloning possible*Presence of selective marker, Ampicillin resistant gene
Plasmid Construction of Arabidopsis Plantacyanin
Primers 1&3
Primer 1: AtPNCNdeMet1 gatcgacatatggccaagggaagaggcagtgc
Primer 3: AtPNCNdeMet32 tacgttcatatggcaacgtacacggtcggtgactctgg
Reverse Primer: AtPNCBamStop tagaggatccatcaaaccgcggtgactgcgattttc
P32
P1
Plasmid Construction of Lily Chemocyanin
Primer 2: lilChemNdeMet1 atctatcatatggctcagggaagtggcagtgcag
Primer 4: lilChemNdeMet30 gtggcccatatggtcgtctataccgtcggcgatggc
Reverse Primer: lilChemBamStop gagaggatccttaagctgctgtgactgctatcttcaaacc
L30
L1
PCR Condition
JRAY
94°C 2min94°C 1min50°C 1min72°C 2min
72°C 10min4°C 99hr
N2
94°C 2min94°C 1min50°C 1min72°C 4min
72°C 10min4°C 99hr
30X
*Reaction program Jray was the first program that I used. Using Jray program yielded product, yet the size of the DNA was incorrect. *Reaction program N2 is 60min longer than Jray and using this program yielded right size DNA
DNA Purification after PCR
Agarose Gel Electrophoresis
Low Melting Agarose GelElectrophoresis
Phenol-Chlorform Extraction Cold Ethanol
Precipitation
•DNA is in aqueous phase in phenol-chloroform extraction•After ethanol precipitation, DNA is in the pallet
DNA Digestion & Ligation
Digest DNA with restriction enzyme, NdeI & BamHI with NEB buffer #2
Purify DNA After Restriction Digestion
DNA Ligation with pET-3a vector
Transformation into Competent Cells
Conclusion
*P1 expression produced large quantities of recombinant protein*No presence of protein for L1*Protein in P1 can be purified via glucose shock then ultrafiltration*Protein in L30 should be purified via sonication, 8M urea treatment, and ultrafiltration
Protein Purification: Ultrafiltration of P1
Protein Purification: L30 purification
Further Research
Purified proteins will be sent to University of California, Riverside for further study of Lily Chemocyanin
Acknowledgement
First of all I want to thank God, Dr. Nersissian, Brooke Vuong, and Nicole. And I also want to thank Sam Phang, Penny Saephan, Phuong Minh and Nersissian Lab folks.
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