Finding the protein interacting with his-tagged Orf1 in M.smegmatis
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Finding the protein interacting with his-tagged Orf1 in M.smegmatis Orf1 Constructing Yeast two hybrid vector of EF-tu Preparing of Nothern blotting (confirming the expression of EF-Tu in 7H9+0.2%glucose or 7H9 30% CO ) Orf2 Transfomation pMsm orf2 vector in BL21 ->Induction using by IPTG (Isopropyl-β-DThioga lactopyranoside) ->Setting the induction condition ->purification of His-tagged Orf2 ->Confirming the his-tagged Orf2 using Western blotting ->Co-immuno-precipitation of the protein inter acting with His-tagged Orf2 from M. smegmatis Re-extract peptide interacting with Orf2 pMsmOrf2 5436 bp bla lacI His-tagged Orf2 orf2 pMsmOrf2-T/NcoI and XbaI Nco I (341) Xba I (1087)
description
Finding the protein interacting with his-tagged Orf1 in M.smegmatis. Orf1 Constructing Yeast two hybrid vector of EF-tu Preparing of Nothern blotting (confirming the expression of EF-Tu in 7H9+0.2%glucose or 7H9 30% CO ). Orf2 Transfomation pMsm orf2 vector in BL21 - PowerPoint PPT Presentation
Transcript of Finding the protein interacting with his-tagged Orf1 in M.smegmatis
Finding the protein interacting with his-tagged Orf1 in M.smegmatis Orf1Constructing Yeast two hybrid vector of EF-tuPreparing of Nothern blotting(confirming the expression of EF-Tu in 7H9+0.2%glucose or 7H9 30% CO )
Orf2Transfomation pMsm orf2 vector in BL21->Induction using by IPTG (Isopropyl-β-DThiogalactopyranoside) ->Setting the induction condition->purification of His-tagged Orf2->Confirming the his-tagged Orf2 using Western blotting ->Co-immuno-precipitation of the protein interacting with His-tagged Orf2 from M. smegmatis Re-extract peptide interacting with Orf2
pMsmOrf25436 bp
bla
lacI His-tagged Orf2
orf2
pMsmOrf2-T/NcoI and XbaI
Nco I (341)
Xba I (1087)
Yeast two hybrid vector of EF-Tu
pGAD GH-EF-Tu9188 bp
Ampicillin resistance gene
EF-Tu
LEU2
Col E1 plasmid replication origin
S. cerevisiae ADH1 terminator
Eco RI (854)
Nco I (1968)
Sma I (844)
Xma I (842)
Bam HI (836)
Bam HI (1465)
Cla I (2052)
Cla I (3783)
Hin dIII (401)
Hin dIII (2402)
Pst I (852)
Pst I (4632)
Pst I (8123)
Apa LI (2215)Apa LI (5190)
Apa LI (6437)
Apa LI (6934)
Apa LI (8733)
Ava I (435)
Ava I (842)
Ava I (1397)
Ava I (1898)
Ava I (2066)
Ava I (7812)
pAS2-1 EF-Tu9567 bp
TRP1 coding sequence
GAL4 binding domain polypeptide
Ampicillin resistance gene
CYH2 gene
EF-Tu
Saccharomyces cerevisiae ADH1 promoter
2¥ì origin of replication
f1 single-strand DNA origin
pUC origin of plasmid replication
CYC1 terminator
Bam HI (6599)
Eco RI (5988)
Pst I (7189)
Nco I (5977)
Nco I (7102)
Apa LI (1590)
Apa LI (2595)
Apa LI (8072)
Apa LI (9318)
Hin dIII (2397)
Hin dIII (3284)
Hin dIII (5477)
Hin dIII (7399)
Ava I (294)
Ava I (3297)
Ava I (3305)
Ava I (5719)
Ava I (6531)
Ava I (7032)
B1 B2 B3 B4 M P1 P2 P3 P4
M:1Kb ladder
P: prey vector->1201bps
B: Bait vector->1201bps
1201bps1201bps
Co-immuno-precipitation of the protein interacting with His-tagged Orf1 & Orf2 from M. smegmatis
174
117
76
51
39
26
Orf1 Orf2
174
117
76
51
39
26
IgGOrf1
Orf2
Purification of his-tagged Orf2 & Western blotting
M C E9 C E9M C FT W E1 E2 E5 E7 E9 E11
174117
7651
39
26
약 28KDa
MALDI-TOF analysis the protein interactiong with Orf1
Streptomyces avermitilis MA-4680 Streptomyces collinus Streptomyces coelicolor A3
Streptomyces aureofaciens Streptomyces netropsis
Streptomyces ramocissimus Streptomyces mobaraensis
Streptomyces ramocissimus Planobispora rosea Thermobifida fusca YX
Acidothermus cellulolyticus 11B Frankia alni str. ACN14A
Frankia sp. CcI3 Micromonospora sp. ATCC 39149
Propionibacterium acnes KPA171202
Corynebacterium glutamicum Corynebacterium efficiens YS-314 Corynebacterium diphtheriae gravis NCTC13129
Corynebacterium jeikeium K411 Rhodococcus sp. RHA1 Nocardia farcinica
Mycobacterium sp. MCS Mycobacterium smegmatis str. MC2
Mycobacterium bovis Mycobacterium tuberculosis
Mycobacterium leprae Mycobacterium ulcerans
Mycobacterium avium Brevibacterium linens Arthrobacter sp. FB24
Micrococcus luteus
10097
100
97
100
100
100
41
54
44
26
99
90
96
63
83
65
54 47
62
33
38
64
9871
72
4535
66
0.02
Corynebacterium glutamicum ATCC 13032
Phylogenetic tree of EF-Tu
Random mutagenesis in M.smegmatis
pTnMod-OKm5098 bp
Tn5 transposase
kanamycin 3'-phosphotransferase
pMB1 narrow-host-range origin of replication
Tn5 inside inverted repeat
Tn5 outside inverted repeat
RP4 origin of transfer
Apa LI (402)
Eco RI (2093)
Sma I (1606)
Xma I (1604)
Cla I (787)
Cla I (1787)
Hin dIII (1358)
Hin dIII (3899)
Pst I (3261)
Pst I (4418)
Ava I (795)
Ava I (1604)
Ava I (1878)
Ava I (2033)
Ava I (4609)
pTnMod-OHyg5164 bp
Tn5 transposase
HygR
Tn5 inverted repeat
ColE1
Tn5 inverted repeat
RP4 origin of transfer
From Klenow filled in pKO/XbaI
Eco RI (1167)
Eco RI (2159)
Sac I (796)
Sac I (1207)
Sac I (2106)
Obtained 7 mutant candidates
pTnSH6399 bp
Tn5 transposase
HygR
SacB
pMB1
Tn5 inside end
Tn5 outside end
RP4 origin of transfer
From pOSH/XbaI
Apa LI (1909)
Bam HI (1489)
Sma I (1497)
Xma I (1495)
Hin dIII (1519)
Hin dIII (5200)
Cla I (732)
Cla I (1069)
Cla I (1526)
Eco RI (1507)
Eco RI (2433)
Eco RI (3394)
Pst I (1505)
Pst I (4562)
Pst I (5719)
Ava I (1495)
Ava I (2250)
Ava I (2472)
Ava I (5910)
