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Finding the protein interacting with his-tagged Orf1 in M.smegmatis

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

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Finding the protein interacting with his-tagged Orf1 in M.smegmatis

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  1. 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-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

  2. Yeast two hybrid vector of EF-Tu

  3. B1 B2 B3 B4 M P1 P2 P3 P4 M:1Kb ladder P: prey vector->1201bps B: Bait vector->1201bps

  4. Bait vector -> performing the sequencing • Prey vector ->performing the sequencing

  5. Co-immuno-precipitation of the protein interacting with His-tagged Orf1 & Orf2 from M. smegmatis Orf1 Orf2 174 174 117 117 76 76 IgG 51 Orf1 51 39 39 26 26 Orf2

  6. Purification of his-tagged Orf2 & Western blotting 174 117 76 51 39 26 약 28KDa M C FT W E1 E2 E5 E7 E9 E11 M C E9 C E9

  7. MALDI-TOF analysis the protein interactiong with Orf1

  8. Streptomyces avermitilis MA-4680 45 35 Streptomyces collinus Streptomyces coelicolor A3 66 Streptomyces aureofaciens 72 Streptomyces netropsis 71 98 Streptomyces ramocissimus 64 Streptomyces mobaraensis 38 Streptomyces ramocissimus 33 Planobispora rosea Thermobifida fusca YX 62 Acidothermus cellulolyticus 11B 54 Frankia alni str.ACN14A 47 Frankia sp. CcI3 100 65 Micromonospora sp. ATCC 39149 Propionibacterium acnes KPA171202 100 97 Corynebacterium glutamicum 97 Corynebacterium efficiens YS-314 100 Corynebacterium diphtheriae gravis NCTC13129 Corynebacterium jeikeium K411 Rhodococcus sp.RHA1 83 Nocardia farcinica 63 Mycobacterium sp.MCS 54 Mycobacterium smegmatis str.MC2 90 Mycobacterium bovis 100 99 Mycobacterium tuberculosis Mycobacterium leprae 26 Mycobacterium ulcerans 44 Mycobacterium avium 41 Brevibacterium linens Arthrobacter sp.FB24 96 Micrococcus luteus 100 0.02 Phylogenetic tree of EF-Tu Corynebacterium glutamicum ATCC 13032

  9. Random mutagenesis in M.smegmatis

  10. Obtained 7 mutant candidates • Confirming the mutation site

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