His pull-down assay for Orf6 in Mycobacterium sp. strain JC1 -> Yeast two hybrid

Study for rv3676-homolog in Mycobacterium sp. strain JC11. Primer extension2. Mutagenesis CO oxidizer Pseudonocaridia was deposit in KCCM Pandoraea 16s rDNA sequencing Tsukamurella

CO-DH expression in M. tuberculosis ? Northern blot or Western blot

Confirmation of transcription level in Mycobacterium sp. strain JC1 (under SNP) cutBCA, orf1-6…

Confirmation of transcription level in Mycobacterium smegmatis (under SNP)

CO-DH expression in Mycobacterium smegmatis

CutR expression pMal C2X vector 이용 -> sequencing 의뢰

Searching the anti-tuberculosis drug from natural compound 흰쌀 , 지골피 , 측백잎 , 황경피나무껍질 , 마늘 , 고삼 , 백급 , 대추 , 의이인

분자설계연구소 200 개의 compound test. test 완료 . XDH inhibitor Allopurinol, oxypurinol, BOF 4272, FYX, Y-700, TEI-6720

CO-DH purification from Mycobacterium smegmatis

Mutagenesis for das gene Searching the single crossover mutant candidates

CO-DH expression in Mycobacterium sp. strain JC1 mod mutant

Mycobacterium smegmatis mc2-155

No cbbLS gene in database has key enzymes for reverse TCA cycle

PCR product for cbbLS gene in Mycobacterium smegmatis mc2-155 wild type, orf2- mutant, and orf4- mutant.

Paper work

1. Mycobacterium sp. strain JC1 CO-DH cloning

Cosmid libraryPrimer extension

2. Mycobacterium sp. strain JC1 RubisCO cloning

Library screening method…

Southern blot for cbbL gene

Confirm : #41, 49, 57 61, 70, 77, 81, 84, 92, 94, 97, 112, 114, 119, 150 ,151, 152, 161, 169, 174, 176

SMB-MeOH

A : CBB stainingB : CO-DH activity staining

Lane 1 : Mycobacterium sp. strain JC1 wild typeLane 2 : Mycobacterium sp. strain JC1 copy I CO-DH deletion mutantLane 2 : Mycobacterium sp. strain JC1 copy II CO-DH deletion mutant

1 2 3 1 2 3

A B

SMB-CO

SMB-glucose0.29 0.27 0.25

27.4 5.0 0.8

507 5.0 427

105.6 91.2 34.6

7.0 8.5 15.6

IRIR22

cutB1

CACGCGCTGAACGTACCCGAGGGCCAGTTAAGCGATTCCTTAAGGGGGGTGTTGAGAGCTCGCTTCGACTGCGTCTACCG

GTGCGCGACTTGCATGGGCTCCCGGTCAATTCGCTAAGGAATTCCCCCCACAACTCTCGAGCGAAGCTGACGCAGATGGC

TCGCAGACCGCGGTGTCGATCGCGGCTGAGATCATCGCGTGCCAGTGGGGTGGTGGCGGGCGTCCGCTGGCCCAGCTCGG

AGCGTCTGGCGCCACAGCTAGCGCCGACTCTAGTAGCGCACGGTCACCCCACCACCGCCCGCAGGCGACCGGGTCGAGCC

TGGCCGCATCCATCACGAGTGGGGGTCTGACGTGGGCAAACGCGGCTCCGATTACGGATCAGACCCCGAGTTAAGTGATT

ACCGGCGTAGGTAGTGCTCACCCCCAGACTGCACCCGTTTGCGCCGAGGCTAATGCCTAGTCTGGGGCTCAATTCACTAA

CCTTAACTCGCTATTGACGCCATCGCGGCGGCAACGGAAACTAGCCACTTCAGCCACGACGTTGTGAGGATGATCACATG

GGAATTGAGCGATAACTGCGGTAGCGCCGCCGTTGCCTTTGATCGGTGAAGTCGGTGCTGCAACACTCCTACTAGTGTAC

cutR +1 (cutR)-10 (cutR) -35 (cutR)

IRIR11

+1 (cutB1)-10 (cutB1)-35 (cutB1)

CBSCBS+1 (cutB1)

-10 (cutB1)-35 (cutB1)

cutB primer extension

GA T C

3’

G

T

T

T

G

C

G

C

C

G

A

5’

3’

G

G

T

G

A

A

G

T

C

G

5’

glucose CO

cutR primer extension

G A TC 1 23’

G

G

G

G

G

A

A

T

T

C

C

T

T

A

G

5’