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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
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.


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


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

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



Sequence homology with some mycobacteria
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
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)


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


Induction of his tagged orf4 with iptg
Induction of his-tagged Orf4 with IPTG in

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


Purification of the co dehydrogenase of mycobacterium sp strain jc1
Purification of the CO dehydrogenase of in 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


4787 bp in

3592 bp

5415 bp

2964 bp


Co-Immuno-Precipitation of the CutR of in 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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