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