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Lab meeting. November 25, 2006

Lab meeting. November 25, 2006. Jae ho, LEE. 1. Detection of proteins interacting with Orf4 and CutR by Yeast Two Hybrid experiment - chromosomal DNA library of Mycobacterium smegmatis as a prey vector - orf4, cutR bait vector.

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Lab meeting. November 25, 2006

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  1. Lab meeting. November 25, 2006 Jae ho, LEE 1.Detection of proteins interacting with Orf4 and CutR by Yeast Two Hybrid experiment - chromosomal DNA library of Mycobacterium smegmatis as a prey vector - orf4, cutRbait vector. - positive control (pGBT9  TRP coding gene pGAD424  LEU coding gene) - colony selection in -Ade/-His/-Leu/-Trp DO/SD with 3-AT(3-amino- 1,2,4-triazole) plate - beta-galactosidase activity assay with oNPG as a substrate - Orf4 : 4 candidates - Plasmid DNA isolation  Transformation of E.coli DH5α with plasmid DNA - CutR : 6 candidates were selected among 60 colonies.

  2. Plasmid DNA isolation of transformed Yeast AH109 Lane 1 : 1 kb ladder Lane 2 : candidate 1-2 Lane 3 : candidate 1-1 Lane 4 : candidate 2-1 Lane 5 : candidate 2-2 Lane 6 : candidate 3-1 Lane 7 : candidate 3-2 Lane 8 : candidate 4-1 Lane 9 : candidate 4-2 1 2 3 4 5 6 7 8 9 10000bp pAS2-1 + orf4 : 9276 bp pGADGH + MSM gDNA library total2 : about 12000~13000 bp

  3. 2.Purification of the CO dehydrogenase of Mycobacterium sp. strain JC1 - Electroporation of the pNBV-1 JC1 cutA(His-tagged) of Mycobacterium sp. strain JC1 cutA mutant Mycobacterium sp. strain JC1 wild type Mycobacterium sp. strain JC1 cutA mutant with pNBV1 JC1 CutA Mycobacterium sp. strain JC1 cutA mutant 3 ml SMB small culture  250 ml large culture carbon source : 30% CO 3 ml SMB small culture  50 ml large culture carbon source : 0.2% Glucose  harvest

  4. Mycobacterium sp. strain JC1 cutA mutant with pNBV1 JC1 CutA 50 ml SMB + 0.2% glucose + hygromycinB  harvest total DNA extraction  transformation of E.coli DH5α 1 2 3 4 5 6 7 8 9 5895 bp 6000 bp 2924 bp 3000 bp Lane 1 : 1 kb ladder Lane 2 : candidate 1 Lane 3 : candidate 2 Lane 4 : candidate 3 Lane 5 : candidate 4 Lane 6 : candidate 1/ClaI Lane 7 : candidate 2/ClaI Lane 8 : candidate 3/ClaI Lane 9 : candidate 4/ClaI pNBV1 JC1 CutA 8819 bp Restriction enzyme reaction by ClaI 5895 bp + 2924 bp

  5. 3. PCR of the homology sequence of hypothetical protein in mycobacterium smegmatis

  6. Sequence homology with some Mycobacteria • hypothetical protein MkmsDRAFT_2415 [Mycobacterium sp. KMS] • hypothetical protein MjlsDRAFT_2401 [Mycobacterium sp. JLS] • hypothetical protein MvanDRAFT_0830 [Mycobacterium vanbaalenii PYR-1]

  7. 5. Purification of the His tagged Rv3676 of Mycobacterium smegmatis Small culture on 7H9 + 0.2% glucose + HygromycinB media Large culture on 7H9 + 30% CO + hygromycinB media 7.5 % Non – denaturing PAGE 1 2 3 4 5 6 7 8 9 10 FIGURE 1 . His tagged Rv3676 purification by Ni-IDA column . lane1 : crude extract, lane2 : wash buffer 1, lane3 : purified crude extract , lane4 : wash buffer 2 lane5 : elution buffer 4, lane6 : elution buffer 6, lane7 : elution buffer 8, lane8 : elution buffer 10, lane9 : elution buffer 12, lane10 : elution buffer 14 Sample loading (sample 20ul + 6X loading dye 4ul)

  8. 6. Detection of protein interacting with Orf4 using His-tag in Mycobacterium smegmatis pMsm orf4 Transformation of E. coli strain BL21 Small culture  large culture on 400ml LB-ampicillin media Induction (0.2 mM IPTG , 18 ℃, 15 hrs) Mycobacterium smegmatis wild type small culture on 4 ml SMB + 0.2% glucose media large culture on 400 ml SMB + 30% CO media

  9. Induction of his-tagged Orf4 with IPTG 1 2 3 4 5 6 7 8 9 10 (kDa) 187 127 80 52 42 27 10 % denaturing polyacrylamide gel Lane 1: protein marker Lane 2: crude extract Lane 3: purified crude extract Lane 4: wash1 Lane 5: elution 3 Lane 6: elution 5 Lane 7: elution 7 Lane 8: elution 9 Lane 9: elution 11 Lane 10: elution 13 35kDa 0.2 mM Isopropyl-β-D-thiogalactoside 18 ℃ , 15 hrs

  10. Purification of the CO dehydrogenase of Mycobacterium sp. strain JC1 Glucose Glucose CO CO 1 2 3 1 2 3 1 2 3 1 2 3 • Figure 1. Western blotting of the CO-DH of Mycobacterium sp. strain JC1 • Mycobacterium sp. strain JC1 wild type • Mycobacterium sp. strain JC1 cutA mutant with pNBV1 JC1 CutA • Mycobacterium sp. strain JC1 cutA mutant competent cell

  11. 4787 bp 3592 bp 5415 bp 2964 bp

  12. Co-Immuno-Precipitation of the CutR of M. smegmatis 1 2 3 FIGURE 3 . Co-Immuno-Precipitation of His tagged MSM CutR protein in 10% SDS gel. (kDa) 187 127 80 52 42 27 Lane 1: protein marker Lane 2: IP crude extract Lane 3: IP product 80 kDa 60 kDa 55 kDa 50 kDa 34 kDa 30 kDa 19 Kda 15 Kda

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