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Purification of Agrobacterium rhizogenes protein (GALLS) required for gene transfer to plants.

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.

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Purification of Agrobacterium rhizogenes protein (GALLS) required for gene transfer to plants.

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  1. Purification of Agrobacterium rhizogenes protein (GALLS) required for gene transfer to plants. By Chris Brown Dr. Walt Ream’s Laboratory Microbiology Department

  2. Background • Agrobacterium damages at least 1.4 million dollars worth of agriculture per year in California and Oregon alone.

  3. Agrobacterium tumefaciens attached to a plant cell. Image by Martha Hawes • Agrobacterium is the only known prokaryote to transfer genes to a eukaryote. • Agrobacterium is used to transfer DNA into plant cells

  4. http://www.nature.com/nature/journal/v433/n7026/images/433583a-f2.2.jpghttp://www.nature.com/nature/journal/v433/n7026/images/433583a-f2.2.jpg http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg • Agrobacterium tumefaciens and Agrobacterium rhizogenes infect wounded plants and transfer plasmid DNA (T-DNA) and virulence (Vir) proteins into plant cells.

  5. GALLS replaces virE2 GALLS replaces virE2 in Agrobacterium tumafaciens Uninfected control virE2-mutant pTi virE2-mutant pTi + GALLS

  6. Map of Galls gene Start Codon GALLS-CT GALLS-FL

  7. 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.

  8. 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.

  9. GALLS Contains ATP-binding, helicase, NLS, and Secretion Signal Domains ATP-binding (Walker A) Wild-type: RASTMVGVAGSAKT K172E: RASTMVGVAGSAET Type IV secretion signal Consensus: RxxxxxxxRxRxRxx GALLS: PKAANDVDRLTRDFDERIRVRGDGRGL ATP-binding (Walker B) Wild-type: RTIGKNTIVVIDE D239N: RTIGKNTIVVINE GALLS-CT Helicase motif III GALLS:KLICVGDDRQLPPVGPGDLL GALLS D: KLIC----------GPGDLL Nuclear Localization Signal GALLS: KRKRAAAKEEIDSRKKMAR TEV: GKKNQKHKLKM(X)31 KRKG

  10. 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.

  11. GALLS-CT Approach • Purify Galls full-length protein 6-His tag Met808Ile

  12. Met808Ile Galls gene Bgl II 6-His-tag pLH 416 pCB 1 Bgl II BglII 1. Restriction Digest with Bgl II 2. Ligation pLH444 Bgl II Bgl II Bgl II Bgl II Bgl II Bgl II Bgl II Bgl II Bgl II Bgl II Bgl II Bgl II

  13. Transformation of plasmid pLH 444 into Escherichia coli DH5α Cell culture, in L-broth E. coli cells were plated on L-agar medium with ampicillin Transformation of pLH 445 into Agrobacterium cells Plasmid DNA extraction from E. coli cells

  14. Purification of GALLS-FL with nickel affinity chromatography Extraction of cells contents through “French press method” Add mixture to column. Discard supernatant solution. Add wash to column. Discard supernatant solution. Add elution buffer to column. Retain supernatant solution. Purified GALLS-FL protein in solution.

  15. 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.

  16. 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

  17. Acknowledgements • HHMI • Dr. Kevin Ahern • Larry Hodges • Dr. Walt Ream

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