Lab meeting november 25 2006
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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

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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.


Lab meeting november 25 2006

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


Lab meeting november 25 2006

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


Lab meeting november 25 2006

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


3 pcr of the homology sequence of hypothetical protein in mycobacterium smegmatis

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


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)


Lab meeting november 25 2006

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

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 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


Lab meeting november 25 2006

4787 bp

3592 bp

5415 bp

2964 bp


Lab meeting november 25 2006

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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