Scribd Logo
Upload

Welcome to Scribd!

  • Rhizogenes Protein (GALLS) : Purification of Agrobacterium Required For Gene Transfer To Plants

    Uploaded by

    Srikant Mishra
    14 views18 pages
    The document summarizes research on purifying the GALLS protein from Agrobacterium rhizogenes, which is required for gene transfer to plants. Key points: - A mutation was introduced to the GALLS gene to abolish translation of the GALLS-CT protein and add a histidine tag for purification of the full-length GALLS-FL protein. - The mutated GALLS gene was inserted into a plasmid and transformed into Agrobacterium cells. - Initial results showed the mutation was incorporated correctly and GALLS-FL protein could be purified using nickel affinity chromatography. - Future experiments are planned to study the DNA binding and helicase activity of the

    Original Description:

    Original Title

    Brown

    Copyright

    © Attribution Non-Commercial (BY-NC)

    Available Formats

    PPT, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document
    The document summarizes research on purifying the GALLS protein from Agrobacterium rhizogenes, which is required for gene transfer to plants. Key points: - A mutation was introduced to the GALLS gene to abolish translation of the GALLS-CT protein and add a histidine tag for purification of the full-length GALLS-FL protein. - The mutated GALLS gene was inserted into a plasmid and transformed into Agrobacterium cells. - Initial results showed the mutation was incorporated correctly and GALLS-FL protein could be purified using nickel affinity chromatography. - Future experiments are planned to study the DNA binding and helicase activity of the

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    Download as ppt, pdf, or txt
    14 views18 pages

    Rhizogenes Protein (GALLS) : Purification of Agrobacterium Required For Gene Transfer To Plants

    Uploaded by

    Srikant Mishra
    The document summarizes research on purifying the GALLS protein from Agrobacterium rhizogenes, which is required for gene transfer to plants. Key points: - A mutation was introduced to the GALLS gene to abolish translation of the GALLS-CT protein and add a histidine tag for purification of the full-length GALLS-FL protein. - The mutated GALLS gene was inserted into a plasmid and transformed into Agrobacterium cells. - Initial results showed the mutation was incorporated correctly and GALLS-FL protein could be purified using nickel affinity chromatography. - Future experiments are planned to study the DNA binding and helicase activity of the

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    Download as ppt, pdf, or txt
    of 18

    Purification of Agrobacterium

    rhizogenes protein (GALLS)


    required for gene transfer to plants.

    By
    Chris Brown
    Dr. Walt Ream’s Laboratory
    Microbiology Department
    Background
    • Agrobacterium damages at least 1.4 million
    dollars worth of agriculture per year in
    California and Oregon alone.
    • Agrobacterium is the only known prokaryote
    to transfer genes to a eukaryote.
    • Agrobacterium is used to transfer DNA into
    plant cells

    Agrobacterium tumefaciens attached to a plant cell.


    Image by Martha Hawes
    • Agrobacterium tumefaciens and Agrobacterium
    rhizogenes infect wounded plants and transfer
    plasmid DNA (T-DNA) and virulence (Vir)
    proteins into plant cells.

    http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg http://www.nature.com/nature/jou
    rnal/v433/n7026/images/433583a
    -f2.2.jpg
    GALLS replaces virE2
    GALLS replaces virE2 in Agrobacterium tumafaciens

    Uninfected control virE2-mutant pTi virE2-mutant pTi + GALLS


    Map of Galls gene

    Start GALLS-CT
    Codon

    GALLS-FL
    Understanding GALLS proteins

    • GALLS-FL and GALLS-CT interact.

    • A mutation in the GALLS-CT start codon from a


    Methanine to an Isolucene abolishes translation
    of GALLS-CT.
    •Methionine to isoleucine mutation on
    the 808th codon of GALLS does not
    affect synthesis or activity of GALLS-FL
    •GALLS-CT is required in some hosts
    and not in others.
    GALLS Contains ATP-binding, helicase, NLS, and
    Secretion Signal Domains

    ATP-binding (Walker A)
    Wild-type: RASTMVGVAGSAKT
    K172E: RASTMVGVAGSAET

    Type IV secretion signal


    ATP-binding (Walker B) Consensus: RxxxxxxxRxRxRxx
    Wild-type: RTIGKNTIVVIDE GALLS: PKAANDVDRLTRDFDERIRVRGDGRGL
    D239N: RTIGKNTIVVINE

    GALLS-CT
    Helicase motif III Nuclear Localization Signal
    GALLS: KLICVGDDRQLPPVGPGDLL GALLS: KRKRAAAKEEIDSRKKMAR
    GALLS : KLIC----------GPGDLL TEV: GKKNQKHKLKM(X)31 KRKG
    Research question: Can you build a mutant Galls
    gene to better purify GALLS-FL protien?

    Hypothesis: Creation of start codon mutation of


    GALLS-CT along with a histidine tag will
    better purify GALLS-FL.
    Approach
    Purify Galls full-length protein

    6-His tag
    Met808Ile

    GALLS-CT
    Galls Met808Ile
    Bgl 1. Restriction
    gene Digest with Bgl II
    II
    6-His-tag pBluescript_II_KS(PLUS)
    (296 1 bp )

    pLH 416
    pBluescript_II_KS(PLUS)

    pCB 1
    (2961 bp)

    pBlueScribe_SK(PLUS)
    pBlueScribe_SK(PLUS)
    (2964bp)
    bp)
    Bgl Bgl II Bgl
    Bgl II

    II
    (2964

    II

    2. Ligation

    pLH444
    Transformation of plasmid pLH
    444 into Escherichia coli DH5α

    Cell culture, in
    E. coli cells were L-broth
    plated on L-agar
    medium with
    ampicillin

    Transformation of pLH 445 into


    Agrobacterium cells Plasmid DNA
    extraction from
    E. coli cells
    Purification of GALLS-FL with nickel affinity chromatography

    Extraction of cells contents through Add mixture to column. Discard


    “French press method”
    supernatant solution.

    Add wash to column. Discard


    supernatant solution.

    Add elution buffer to column.


    Retain supernatant solution.

    Purified GALLS-FL protein in solution.


    Results obtained along with Larry
    Hodges
    • Ligated Bgl II fragment containing Met808Ile
    mutation into 6-His tagged Galls.
    – Correct orientation of the ligated fragment
    • Ligated His-Galls gene into Agrobacterium
    plasmid and transformed into Agrobacterium.
    • Cultured Agrobacterium cells for purification
    of GALLS-FL protein.
    Future experiments
    • DNA binding assays
    – Determine if GALLS-FL binds to single stranded
    DNA or double stranded DNA.
    • Helicase assay to determine type of DNA used
    by GALLS-FL
    • ATP binding and hydrolysis assays
    Acknowledgements
    • HHMI
    • Dr. Kevin Ahern
    • Larry Hodges
    • Dr. Walt Ream

    You might also like