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