Presentation on theme: "Purification of Agrobacterium rhizogenes protein (GALLS) required for gene transfer to plants. By Chris Brown Dr. Walt Ream’s Laboratory Microbiology Department."— Presentation transcript:
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://www.nature.com/nature/jour nal/v433/n7026/images/433583a- f2.2.jpg http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg
GALLS replaces virE2 Uninfected control virE2-mutant pTi virE2-mutant pTi + GALLS GALLS replaces virE2 in Agrobacterium tumafaciens
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. Understanding GALLS proteins
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 ATP-binding (Walker B) Wild-type: RTIGKNTIVVIDE D239N: RTIGKNTIVVINE Helicase motif III GALLS: KLICVGDDRQLPPVGPGDLL GALLS : KLIC----------GPGDLL Nuclear Localization Signal GALLS: KRKRAAAKEEIDSRKKMAR TEV: GKKNQKHKLKM(X) 31 KRKG Type IV secretion signal Consensus: RxxxxxxxRxRxRxx GALLS: PKAANDVDRLTRDFDERIRVRGDGRGL GALLS-CT
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 GALLS-CT Met808Ile 6-His tag
Bgl II 1. Restriction Digest with Bgl II 2. Ligation 6-His-tag pCB 1 Bgl II pLH444 Met808Ile pLH 416 Bgl II Galls gene
Transformation of plasmid pLH 444 into Escherichia coli DH5α E. coli cells were plated on L-agar medium with ampicillin Cell culture, in L-broth Plasmid DNA extraction from E. coli cells Transformation of pLH 445 into Agrobacterium cells
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. Purification of GALLS-FL with nickel affinity chromatography 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