lab meeting. november 25, 2006
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
Lab meeting. November 25, 2006
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.
- 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.
Jae ho, LEE
Plasmid DNA isolation of transformed Yeast AH109
1 2 3 4 5 6 7 8 9
10000bp
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
pAS2-1 + orf4 : 9276 bp
pGADGH + MSM gDNA library total2 : about 12000~13000 bp
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
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
3000 bp 2924 bp
5895 bp6000 bp
pNBV1 JC1 CutA 8819 bp
Restriction enzyme reaction by ClaI 5895 bp + 2924 bp
Lane 1 : 1 kb ladder
Lane 2 : candidate 1Lane 3 : candidate 2Lane 4 : candidate 3Lane 5 : candidate 4Lane 6 : candidate 1/ClaILane 7 : candidate 2/ClaILane 8 : candidate 3/ClaILane 9 : candidate 4/ClaI
3. PCR of the homology sequence of hypothetical protein in mycobacterium smegmatis
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]
5. Purification of the His tagged Rv3676 of Mycobacterium smegmatis
Small culture on 7H9 + 0.2% glucose + HygromycinB mediaLarge culture on 7H9 + 30% CO + hygromycinB media
1 2 3 4 5 6 7 8 9 10
7.5 % Non – denaturing PAGE
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)
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
pMsmOrf45595 bp
bla
lacI His-tagged Orf4
orf4
pMsmOrf4-T/NcoI and XbaI
Nco I (341)
Xba I (1246)
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 markerLane 2: crude extract Lane 3: purified crude extractLane 4: wash1Lane 5: elution 3Lane 6: elution 5Lane 7: elution 7Lane 8: elution 9Lane 9: elution 11Lane 10: elution 13
0.2 mM Isopropyl-β-D-thiogalactoside 18 , 15 hrs ℃
35kDa
Purification of the CO dehydrogenase of Mycobacterium sp. strain JC1
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
1. Mycobacterium sp. strain JC1 wild type
2. Mycobacterium sp. strain JC1 cutA mutant with pNBV1 JC1 CutA
3. Mycobacterium sp. strain JC1 cutA mutant competent cell
CO Glucose Glucose CO
pNBV1/EcoR¥´ + pMsmOrf4/Sph¥°8379 bp
HygR
pAL5000
pMsmOrf4/sph¥°
Nco I (445)
Nco I (4037)
pNBV1/EcoR¥´ + pMsmOrf4/SphI reverse8379 bp
HygR
pAL5000
reverse pMSMorf4
Nco I (445)
Nco I (3409)
4787 bp
3592 bp
5415 bp
2964 bp
Co-Immuno-Precipitation of the CutR of M. smegmatis
(kDa)
187
127
80
52
42
27
Lane 1: protein marker Lane 2: IP crude extract Lane 3: IP product
FIGURE 3 . Co-Immuno-Precipitation of His tagged MSM CutR protein
in 10% SDS gel.
80 kDa
55 kDa
19 Kda
15 Kda
1 2 3
60 kDa
50 kDa
34 kDa
30 kDa