Fluorescent Assay of a RING-type Ubiquitin Ligase

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Fluorescent Assay of a RING-type Ubiquitin Ligase
Mississippi State University November 2007
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Our Team
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Our Team - Students

• Agricultural and Biological Engineering
• Graduate
• Robert Morris
• Undergraduate
• Lauren Beatty, Scott Tran, Joe Chen, Caleb
  Dulaney, Sam Pote, Karen Parks
• Biochemistry
• Graduate
• Victor Ho
• Undergraduate
• James Kastrantas

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Our Team - Professors

• Agricultural and Biological Engineering
• Dr. Filip To
• Biochemistry
• Dr. Din-Pow Ma
• Electrical and Computer Engineering
• Dr. Bob Reese
• Chemical Engineering
• Dr. Todd French

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Present Areas of Interest

• Departmental Goal
• Discover lipid production pathways
• Increase lipid synthesis in plants
• Make biofuels more economically feasible
• iGEM 2007 Goal
• Design a construction that simplifies and
  expedites the confirmation of ubiquitin ligase
  activity

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Ubiquitin Proteasome Pathway

• The ubiquitination of target proteins for
  degradation requires the sequential activity of
  three enzymes
• E1, ubiquitin activating enzyme
• E2, ubiquitin conjugating enzyme
• E3, ubiquitin ligase

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Ubiquitin Proteasome Pathway
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Methodology

• A RING-type E3 gene, GhR1(1kb), was amplified by
  PCR and cloned into pGFPuv as an in-frame fusion
  at N-terminal with GFPuv.
• Chemically competent (CaCl2) E. coli XL1-blue
  cells were transformed with recombinant plasmid
  pGFP/GhR1.
• Transformed XL1-blue cells were grown to OD600
  0.6 and lysed by sonnication.

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Methodology

• Cell lysate was added to ubiquitin and wheat germ
  extract, which provides reagents (E1, E2, ATP)
  for ubiquitination.
• Reaction mixtures were analyzed by native PAGE.
• Excitation of the fusions revealed the presence
  of E3 and ubiquitination on the gel.

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Project Justification

• Benefits
• Assays E3 ligase activity directly on native
  protein gel
• Omits primary purification step, Western blot,
  and pull down assay
• Saves time and resources

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Desired Machine Function

• Rapidly report ubiquitin ligase activity via
  green fluorescence

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Initial Idea for Plasmid Construction
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Initial Idea for Plasmid Construction
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Initial Idea for Plasmid Construction
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Problems Encountered

• Upon inspection of our construction plans
  involving the insertion of ubiquitin next to the
  Registry part J01095, the S/X restriction site
  created with the ligation of these two parts
  would code for the stop codon UAG.

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First Plasmid Construction
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Confirmation of Constructions

• All constructions were sequenced using ABI 310
  Prism Genetic Analyzer.
• All sequences were confirmed to be correct and in
  frame with the GFP gene.

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Results of First Construction

• Lane ID
• 1 Protein Ladder
• 2 GhR1/GFP
• 3 GFP
• 4 GhR1/GFP/Ub
• 5 GFP/Ub

1 2 3 4 5
1 2 3 4 5

Ub Ubiquitin
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Analysis of First Construction

• The GhR1-GFP fusion protein in lanes 2 and 4 had
  fluorescence.
• No multiple protein bands were detected in lane 4
  after Western blotting with anti-ubiquitin,
  suggesting that ubiquitination did not occur.

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Second Plasmid Construction
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Results of Second Construction

• Lane ID
• 1 Protein Ladder
• 2 Sh-GhR1/GFP
• 3 Fl- GhR1/GFP
• 45 GFP

1 2 3 4 5

Sh short Fl full
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Analysis of Second Construction

• GFP without the fusion in lanes 4 and 5 showed
  fluorescence.
• There was no visible fluorescence in lanes 2 and
  3, suggesting that the expression level of
  GhR1-GFP fusion was either too low or the fusion
  protein changed conformation with no
  fluorescence.

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Conclusions

• The first construction was successful with the
  observation of fluorescence from the GhR1-GFP
  fusion protein.
• The first construction failed to detect
  poly-ubiquitin chains.
• GFP adjacent to the RING domain (C-terminal) of
  GhR1 might block ubiquitination.

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Conclusions

• There was no expression of green fluorescence in
  the second construction.
• Expression of the GhR1-GFP fusion was not
  observed in either the short or full length form
  of GhR1.
• This could be the result of an alteration of
  protein structure from the fusion of GhR1 with
  GFP.

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Future Work

• Fusion of RFP with GhR1
• One single plasmid for both GFP-E3 and
  RFP-ubiquitin fusions
• Discovery of other protein-protein interactions
• Interactions controlling lipid synthesis

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Future Work

• Understanding of the regulation of lipid
  production will enable the design of new machines
  with predetermined lipid content
• Higher lipid content in plant ? Higher energy
  value of biofuel
• Lower undesired lipid levels ? Higher product
  value
• May lead to understanding of other regulatory
  pathways (polysaccharides)

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Acknowledgements

• Advisors
• Dr. Filip To, Dr. Din-Pow Ma
• MSU Bagley College of Engineering
• MSU College of Agriculture and Life Sciences
• USDA Strategic Research Initiative
• iGEM Staff

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• Questions?