Arabidopsis thaliana ORFeome Project

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Arabidopsis thaliana ORFeome Project

• By
• Raquel Sojourner

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What is Arabidopsis thaliana and why do we use it?

• Comes from the Mustard Seed family.
• Only 30,000 genes in its genome.
• Can be planted, grown up, and produce seeds in
  2-3 months.

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What is the ORFeome project?

• A Project with a goal to get every gene in the
  Arabidopsis genome represented as an ORF and as a
  UTR clone.
• Then make the clones available to the entire
  plant biology community.

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What is an ORF?

• Open-Reading-Frame
• This open reading frame has a start and stop
  codon and codes for protein expression.
• Making an ORFclone for every gene allows for the
  study of functional genomics and proteomics .

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What is a UTR?

• UnTranslated-Region
• Located on the 5 prime and 3 prime ends of the
  ORF.
• The untranslated region of the ORFclone has the
  sequence on how to make the protein.
• All UTRclones eventually become ORFclones.

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How do you make these clones?

• In order to get a complete ORF clone or UTR clone
  it takes a series of processes.
• PCR- amplifying the DNA.
• DIGEST- cutting the PCR product for the vector.
• LIGATION- gluing the PCR product to the vector
  in order to form a circular plasmid.
• TRANSFORMATION- inserting the plasmid into the
  bacteria.
• PLASMID PREP- extracting the plasmid from the
  bacteria.
• SEQUENCING- sequence verifying that an actual
  clone was made.

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PCR

• Polymerase-Chain-Reaction
• Amplification (multiplying the gene)
• The template we use to amplify from is Reverse
  Transcription which comes from mRNA from the
  tissues of the Arabidopsis or DNA from UTRclones.
• Gene specific primers are put into the DNA to
  isolate a specific region on the DNA.
• These primers also have internal sfi sites that
  become part of the DNA when they are attached.

• Three Steps
• 1) Denaturation- raising the temperature to 96
  degrees Celsius to separate the DNA into two
  strands.
• 2) Annealing- primer recognition of the single
  strand DNA at 55 degrees Celsius.
• 3) Extension- extaq enzyme is used to extend
  strand at 72 degrees Celsius.

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Digest

• The restriction enzyme, Sfi 1, cuts the the two
  Sfi sites at each sfi site of the PCR product.

• This prepares the amplified gene to be ligated or
  glued to the vector.

Sfi 1 cuts the gene at the arrows.
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Ligation

• The Sfi 1 ends of the gene are glued to the
  sticky Sfi 1 sites of the vector with T4 DNA
  ligase.
• When glued together they become a circular
  plasmid which enables them to be inserted into
  the bacterial host.

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Puni51Vector

• Two sticky Sfi 1 site ends.
• R6K? site where replication happens.
• KAN site which resists the Kanamycin in the
  growth media.

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Transformation

• Plasmid inserted into E. coli bacterial host.
• During replication it will also replicate the
  plasmid with the gene.
• Spread onto an LB agar dish and grown up
  overnight.
• If bacteria colonies are produced, they are
  picked and grown in TB media.

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Plasmid Prep/Sequencing

• The plasmid is extracted from the bacteria.
• Plasmid is sequence verified to ensure that an
  actual clone was made without mutations.
• Plasmid could just contain a vector without a
  gene, a fragment of a gene or a mutated gene.

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Project 244

• 244 goal- UTRclones
• Started with about 46 genes.
• Used RT and repooled primers.
• Trouble with amplification.
• Total of 4 PCRs.
• Results came with a change in RT.
• Genes lost in PCR, Digest and Transformation.
• Ended with about 16 UTR clones.
• 33 cloned

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Project 307

• 307- goal ORFclones
• Started with 32 genes.
• Diluted primers.
• Used repooled DNA.
• Great amplification in PCR.
• Genes lost in PCR and Transformation.
• Ended with about 19 ORFclones.
• 60 cloned

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What is done with the clones?

• Distributed to ABRC (Arabidopsis Biological
  Resource Center)
• -depository for seeds, clones, and everything
  that has to do with the Arabidopsis plant and
  makes it available to the plant biology
  community.

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Acknowledgments

• Dr. Joe Ecker
• Christopher Kim
• Rosa Cheuk
• Paul Shinn
• Huaming Chen
• Larry Nordell

• Natalie Chan
• Stephanie Mah
• Heeran Abawi
• Jonathan Ecker
• Ingrid Johnson

Thank You !!!