Acquired Tolerance to Oxygen Stress in Bifidobacterium longum Title 105-A by Heterologous Expression of Catalase Gene( 本文 （FULLTEXT) ) Author(s) 賀, 建龍 Report No.(Doctoral Degree) 博士(農学) 甲第583号 Issue Date 2012-03-13 Type 博士論文 Version publisher URL http://hdl.handle.net/20.500.12099/42969 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。 Contents 1. Introduction---I---------------------------------------------- 1 1.1 Bljidobacterium-------------------------------------------- 1 1.1.1 Description------------------------------------------------1 1.1.2 History-----------------------------------------------4 1.1.3 Species------------------------'-------I-----------------5 1.1.4 Ecology--------------------------------------------------6 1.2 Peroxidase--------------------------------------------9 1.2.1 Description----------i--i---------------------------9 1.2.2 GPX------------------------i-------------------------------10 1.2.3 Catalase------------------------------J-------------------12 1.2.3. 1 Molecular mechanism------------------------------------1 3 1.2.3.2 Cellular role-------------------------------------------------14 1.2.3.3 Distribution among organisms----------------------------------1 5 1.3 Research the response of the Bljidobacterium to Oxygen--------------1 7 2. Materials and Methods---------------------------------------20 2. 1 Media and Buffer------------------------i----------------20 2. 1.1 Luria Broth media-------------------------------------------------20 2.1.2 MRS media-------------------------------------------,-----------20 2.1.3 TE buffer-------------------I-----------------------------21 2.1.4 PEG---------------------------------:-------------------------21 2. 1.5 PBS buffer--I---------------------------------------------------21 2.2 Isolate DNA from microorganisms------------------------------2 1 2.3 Extract plasmid----------------------------------------------2 1 2.3.1 With QIA prep spin Mini prep kit------------------------------------21 2.3.2 With 2-propano1-------------------------------------------22 2.3.2. 1 The reagent--------------.-----------------------------------22 2.3.2.1. 1 Solution I----------------------------------------------22 2.3.2. 1.2 Solution II---------------------------------------------22 2.3.2. 1.3 Solution III----------------------------------------------22 2.3.2.2 Protocol----------------------------------------------------------22 2.4 Purify DNA---------------------------------------23 2.4.1 With Nucleo Spin Extract II kit-------------------------------24 2.4.2 With Ethano1----------------------------------------------------24 I 2.4.2. 1 The reagent-----------------------------------------------24 2.4.2.2 Protocol--------------------------------------------------------------24 I 2.4.3 With PEG--------------------------------------------------------25 2.4.3. 1 The reagent-------------------------------------------------25 2.4.3.2 Protocol--------------------------------------------25 2.5 Preparation of competent cell----------------------------------25 2.5.1 Electrocompetent cell of BiBdobacterium-----------------------25 2.5. 1.1 The reagent-------------------------------------------------25 2.5.1.2 Protocol-------------------------------------------------------------26 2.5.2 Chemically competent cell of E.boli-----------------------------26 2.5.2. 1 The reagent----------------------------------------------------26 2.5.2.2 Protocol------------------------------------------------------27 2.6 SDS-PAGE-----------------------------i----------------------27 2.6.1 The reagent---------------------------------------------27 2.6.2 Protocol--------------------------------------------i-I--29 2.7 Detection activity of catalase--------------------------------------32 2.7.1 Add H202 detect activity.f catalas;----------------i---------32 2.7.1.1 The the.,y-;---------------------i----------------32 2.7. 1.2 Protocol-------------------------------------.---------------32 2.7.2 Assay ofcatalase--------------------------------------32 2.7.2. 1 The reagent----------------------------------------33 2.7.2.2 Protocol------------------------------------------------------------33 2.8 Short-term H202 exposure-------------------------------------------34 2.8.1 The reagent----------------------------------------34 2.8.2 Protocol-------------------------------------------------------------34 2.9 Long-term with aerated cultures-----------------------------------35 2.9.1 The reagent-------------------------------------------35 2.9.2 Protocol---I""_"___"__"______"__""_MM___M""__"Mum""__"__35 2. 10 PCR----------------------------------------------------------3 5 2.10.1 With po1ymerase KOD-PLUS----------------i--------------35 2.10.2 With po1ymerase GO Taq---------------------------------------35 2. 1 1 DNA extraction from qgarose gels-----------------------------36 2.12 Digestion with restriction enzyme----------------------T-----------36 2. 12. 1 The restricti.n enzyme----------------------------------J-i-------36 2. 12.2 Protocol-------------------------------------------------------36 2. 13 DNA ligation--------------------------------------I------36 2. 14 Transformation-----------------------------------------------36 2. 14.1 Chemical transformation---------------------------------------------37 2. 14. 1.1 The reagent-------------------------------------------------37 2. 14. 1.2 Protocol-----------------------------------------------------37 II 2. 14.2 Electroporation---------------------------------------------38 2. 14.2. 1 The reagent-----;-------------------------------------------38 2. 14.2.2 Protocol----------------------------------------------i--38 2. 1 5 Gateway system--------------------------------------------------3 8 2.16 Flow cytometry------------------------------------------39 2. 16.1 The reagent----------------------------------.----------39 2. 16.2 The instrument------------------------------------------------------------39 2. 17 Isolate RNA from microorganisms---------------------I--------------39 2. 18 RT-PCR------------------------------------------------------------------39 2.19 qRT-PCR-------------------------------------------39 2.19.1 The reagent----------------------------------------------39 2. 19.2 The instrument-----------------------------------------------------39 2.20 Assay of H202------------------------------------------40 2.20. 1 The reagent----------------i---------------------------40 2.20.2 Protocol------------------------------------------------------------40 2.2 1 Site-Directed Mutagenesis-------------------------------------40 2.22 The strains, the plasmids and primers-------------------------------42 3. Results---------------------------------------------------------44 3.1 Isolate DNA &om microorganisms--------i--------------------44 3.2 Get the PCR product----------------------------------------------45 3.2.1 Design the prim'ers-------------------------------------------------45 3.2.2 PCR-----------------------------------------------------------45 3.2.2. 1 PCR of Hup-promoter---------------------------------45 3.2.2.2 PCR ofB-KatE--------------------------------------i_"MUM"___"__47 3.2.2.3 PCR of Hup-terminator--------------------------------48 3.2.2.4 Overlap PCR----------------------------------------------49 3.3 Construct destination vector----------------------------------------50 3.3.1 Digest pKKT427----------------------------------------50 3.3.2 Construct destination vector pGUOO1--------------------------51 3.4 Construct the plasmids--------------------------------------53 3.4. 1 BP reaction-------------------------------------------------53 3.4.2 LR ,eacti.n----------------_M"_"_"____""""q_____"_:MM____56 3.5 Analyze the sequences------------------------------------57 3.5.1 Hup-promoter----------------------------------------------58 3.5.2 Hup-terminator---------------------------------------58 3.5.3 Catalase gene--------------------------------------------58 III 3.6 The catalase activity in E. coli UM255---------------------------58 3.7 The catalase activity in B. longum 1 05-A--.--------------------------59 3.7.1 pBCATOO1 (Heme-catalase)activity-----------------------------59 3.8 Short-term H202 exposure OfB.longum 105-A---------------------63 3.9 Long-term with aerated cultures ofB.longum 105-A-i---------------64 3.9. 1 0D-------------------------------------------------------------64 3.9.2 CFU------------------------------------------------------65 3.9.3 LIVE/DEAD----------------------------------------------------------66 3.9.4 H202 accumulation-------------------------------------------67 4. Discussion----------------------------------------------------68 4. 1 Protect Bljidobacterium from oxidative stress by expression of catalase---68 4.2 Improvement of catalase expression level------------------------7 1 4.2.1 Transcription--------------------------------------------------71 4.2.2 Translation-------------------------------------------------------75 5. Conclusion---------------------------------------------------------85 6. References--------------------------------------------------------89 7. Acknowledgments---------------------------------- 1 0 1 IV Intro duction 1.1 Bljidobacterium 1.1.1- Description At birth, the gastrointestinal tract is sterile and incapable of digesting food. Within hours, bacteria ingested during the birthing process rapidly colonize the gut. The as bacteria as are gastrointestinal tract soon contains about 1 0 times many there cells in the body. Hundreds of species are present, many of which are uncultivable and for remain unidentified.