Lab meeting. December 9, 2006

1. Lab meeting. December 9, 2006 Jae ho, LEE 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]

2. 1. PCR of the homology sequence of hypothetical protein in Mycobacterium smegmatis

3. 2. Expression of the His tagged Rv3676 of Mycobacterium smegmatis Small culture on 4ml 7H9 + 0.2% glucose + HygromycinB media  Large culture on 200 ml 7H9 + 30% CO + hygromycinB media Small culture on 4ml 7H9 + 0.2% glucose + HygromycinB media  Large culture on 200 ml 7H9 + 0.2% glucose + hygromycinB media 3. Expression of the His tagged CutR of Mycobacterium smegmatis Small culture on 4ml 7H9 + 0.2% glucose + HygromycinB media

4. 4. 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 condition 10 ml LB-ampicillin media 0.2 mM IPTG (Isopropyl-β-D-thiogalactoside) 37 ℃ for 2 hrs , 18 ℃ for 12 hrs ~ 17 hrs 0.5 mM IPTG 37 ℃ for 3 hrs , 18 ℃ for 12 hrs ~ 16 hrs Mycobacterium smegmatis wild type small culture on 4 ml SMB + 0.2% glucose media large culture on 400 ml SMB + 30% CO media

5. Induction of his-tagged Orf4 with IPTG 1 2 3 4 5 6 7 8 10% denaturing polyacrylamide gel 0.2 mM IPTG , 37 ℃ for 2 hrs , 18 ℃ for 12 hrs ~ 17 hrs Lane 1: protein marker Lane 2: crude extract before treatment of IPTG Lane 3: crude extract after treatment of IPTG; 12 hrs Lane 4: crude extract after treatment of IPTG; 13 hrs Lane 5: crude extract after treatment of IPTG; 14 hrs Lane 6: crude extract after treatment of IPTG; 15 hrs Lane 7: crude extract after treatment of IPTG; 16 hrs Lane 8: crude extract after treatment of IPTG; 17 hrs (kDa) 187 127 80 52 42 27 33.5 kDa : His-tagged Orf4

6. Induction of his-tagged Orf4 with IPTG 1 2 3 4 5 6 7 (kDa) 187 127 80 52 42 27 10% denaturing polyacrylamide gel 0.5 mM IPTG , 37 ℃ for 3 hrs , 18 ℃ for 12 hrs ~ 16 hrs Lane 1: protein marker Lane 2: crude extract before treatment of IPTG Lane 3: crude extract after treatment of IPTG; 12 hrs Lane 4: crude extract after treatment of IPTG; 13 hrs Lane 5: crude extract after treatment of IPTG; 14 hrs Lane 6: crude extract after treatment of IPTG; 15 hrs Lane 7: crude extract after treatment of IPTG; 16 hrs 33.5 kDa : His-tagged Orf4

8. Induction of his-tagged Orf4 with IPTG 1 2 3 (kDa) 187 127 80 52 42 27 10% denaturing polyacrylamide gel Lane 1: protein marker Lane 2: crude extract before treatment of IPTG Lane 3: crude extract of after treatment of IPTG 33.5 kDa 0.2 mM Isopropyl-β-D-thiogalactoside 18 ℃ , 14 hrs

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

15. 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

16. 4787 bp 3592 bp 5415 bp 2964 bp

17. 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