Team:Paris Saclay/Labwork

From 2013.igem.org

(Difference between revisions)
(→Characterize BBa_K1155006)
(→BBa_K1155007)
 
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08/07/13
08/07/13
*  Sequensis analysis of BBa_K1155003 [File:Psright.png|20px]]
*  Sequensis analysis of BBa_K1155003 [File:Psright.png|20px]]
 +
 +
'''WE OBTAIN OUR BIOBRICK.'''
=='''BBa_K1155004'''==
=='''BBa_K1155004'''==
Line 205: Line 207:
07/26/13
07/26/13
* Gel Electrophoresis  to check the correct amplification of PnirB [[File:Pswrong.png|20px]]
* Gel Electrophoresis  to check the correct amplification of PnirB [[File:Pswrong.png|20px]]
-
* PCR of NirB
+
* PCR of PnirB
-
* Electrophoresis of PCR products : NirB [[File:Psright.png|20px]]
+
* Electrophoresis of PCR products : PnirB [[File:Psright.png|20px]]
-
* Gel purification of PCR products : NarK [[File:Psright.png|20px]]
+
* Gel purification of PCR products : PnirB [[File:Psright.png|20px]]
-
* Digestion of PCR products : NarK by EcoRI/PstI
+
* Digestion of PCR products : PnirB by EcoRI/PstI
07/30/13
07/30/13
-
* Denaturation of EcoRI/SpeI used of the digestion of PCR products : NirB
+
* Denaturation of EcoRI/SpeI used of the digestion of PCR products : PnirB
07/31/13
07/31/13
* Digestion of PSB1C3 by EcoRI/PstI'
* Digestion of PSB1C3 by EcoRI/PstI'
* Denaturation of EcoRI/PstI used for the digestion of PSB1C3
* Denaturation of EcoRI/PstI used for the digestion of PSB1C3
-
* Ligation of NirB in PSB1C3
+
* Ligation of PnirB in pSB1C3
-
* Electrophoresis to check the ligation of NirB in PSB1C3 [[File:Psright.png|20px]]
+
* Electrophoresis to check the ligation of PnirB in pSB1C3 [[File:Psright.png|20px]]
* Transformation of BBa_K1155004 [[File:Psright.png|20px]]
* Transformation of BBa_K1155004 [[File:Psright.png|20px]]
Line 235: Line 237:
07/25/13
07/25/13
-
* Colony PCR of nag
+
* Colony PCR of PnarG
-
* Electrophoresis  to check the colony PCR of NarG [[File:Pswrong.png|20px]]
+
* Electrophoresis  to check the colony PCR of PnarG [[File:Pswrong.png|20px]]
-
* Colony PCR of NarG
+
* Colony PCR of PnarG
07/26/13
07/26/13
-
* Electrophoresis of PCR products : NarG [[File:Pswrong.png|20px]]
+
* Electrophoresis of PCR products : PnarG [[File:Pswrong.png|20px]]
-
* PCR of NarG
+
* PCR of PnarG
-
* Electrophoresis of PCR products : NarG [[File:Psright.png|20px]]
+
* Electrophoresis of PCR products : PnarG [[File:Psright.png|20px]]
-
* Gel purification of PCR products : NarG [[File:Psright.png|20px]]
+
* Gel purification of PCR products : PnarG [[File:Psright.png|20px]]
-
* Digestion of PCR products : NarG by EcoRI/PstI
+
* Digestion of PCR products : PnarG by EcoRI/PstI
07/30/13
07/30/13
-
* Denaturation of EcoRI/SpeI used of the digestion of PCR products : NarG
+
* Denaturation of EcoRI/SpeI used of the digestion of PCR products : PnarG
07/31/13
07/31/13
-
* Digestion of PSB1C3 by EcoRI/PstI'
+
* Digestion of pSB1C3 by EcoRI/PstI'
-
* Denaturation of EcoRI/PstI used for the digestion of PSB1C3
+
* Denaturation of EcoRI/PstI used for the digestion of pSB1C3
-
* Ligation of NarG in PSB1C3
+
* Ligation of PnarG in pSB1C3
-
* Electrophoresis to check the ligation of NarG in PSB1C3 [[File:Psright.png|20px]]
+
* Electrophoresis to check the ligation of PnarG in pSB1C3 [[File:Psright.png|20px]]
* Transformation of BBa_K1155005 [[File:Psright.png|20px]]
* Transformation of BBa_K1155005 [[File:Psright.png|20px]]
Line 271: Line 273:
07/25/13
07/25/13
-
* Colony PCR of NarK
+
* Colony PCR of PnarK
-
* Electrophoresis  to check the colony PCR of NarK [[File:Pswrong.png|20px]]
+
* Electrophoresis  to check the colony PCR of PnarK [[File:Pswrong.png|20px]]
-
* Colony PCR of NarK
+
* Colony PCR of PnarK
07/26/13
07/26/13
-
* Electrophoresis of PCR products : NarK [[File:Pswrong.png|20px]]
+
* Electrophoresis of PCR products : PnarK [[File:Pswrong.png|20px]]
-
* PCR of NarK
+
* PCR of PnarK
-
* Electrophoresis of PCR products : NarK [[File:Psright.png|20px]]
+
* Electrophoresis of PCR products : PnarK [[File:Psright.png|20px]]
-
* Gel purification of PCR products : NirB [[File:Psright.png|20px]]
+
* Gel purification of PCR products : PnarK [[File:Psright.png|20px]]
-
* Digestion of PCR products : NirB by EcoRI/PstI
+
* Digestion of PCR products : PnarK by EcoRI/PstI
07/30/13
07/30/13
-
* Denaturation of EcoRI/SpeI used of the digestion of PCR products : NarK
+
* Denaturation of EcoRI/SpeI used of the digestion of PCR products : PnarK
07/31/13
07/31/13
-
* Digestion of PSB1C3 by EcoRI/PstI'
+
* Digestion of pSB1C3 by EcoRI/PstI'
-
* Denaturation of EcoRI/PstI used for the digestion of PSB1C3
+
* Denaturation of EcoRI/PstI used for the digestion of pSB1C3
-
* Ligation of NarK in PSB1C3
+
* Ligation of PnarK in pSB1C3
-
* Electrophoresis to check the ligation of NarK in PSB1C3 [[File:Psright.png|20px]]
+
* Electrophoresis to check the ligation of PnarK in pSB1C3 [[File:Psright.png|20px]]
* Transformation of BBa_K1155006 [[File:Psright.png|20px]]
* Transformation of BBa_K1155006 [[File:Psright.png|20px]]
Line 364: Line 366:
07/30/13
07/30/13
* Digestion of BBa_I732017 by EcoRI/PstI
* Digestion of BBa_I732017 by EcoRI/PstI
-
* Electrophoresis to check the digestion of BBa_I732017 by EcoRI/PstI [[File:Psright.png|20px]]
+
* Digestion of BBa_I732017 by EcoRI/SpeI
-
* Gel purification of digestion of BBa_I732017 by EcoRI/PstI [[File:Psright.png|20px]]
+
* Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI
-
* Digestion of BBa_I732017 by EcoRI/PstI
+
* Gel purification of digestion of BBa_I732017 by EcoRI/SpeI [[File:Pswrong.png|20px]]
-
* Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI [[File:Psright.png|20px]]
+
-
* Gel purification of digestion of BBa_I732017 by EcoRI/PstI [[File:Pswrong.png|20px]]
+
07/31/13
07/31/13
Line 382: Line 382:
* Digestion of BBa_I732017 by EcoRI/SpeI
* Digestion of BBa_I732017 by EcoRI/SpeI
* Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI [[File:Psright.png|20px]]
* Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI [[File:Psright.png|20px]]
-
* PCR of PSB1C3
+
* PCR of pSB1C3
-
* Electrophoresis of PCR of PSB1C3 [[File:Psright.png|20px]]
+
* Electrophoresis of PCR of pSB1C3 [[File:Psright.png|20px]]
-
* Gel purification of electrophoresis of PCR of PSB1C3 [[File:Psright.png|20px]]
+
* Gel purification of electrophoresis of PCR of pSB1C3 [[File:Psright.png|20px]]
08/06/13
08/06/13
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08/07/13
08/07/13
-
* Ligation of RBS-LacZ-Term in PSB1C3
+
* Ligation of RBS-LacZ-Term in pSB1C3
* Tranformation BBa_K115507 in DH5α [[File:Psright.png|20px]]
* Tranformation BBa_K115507 in DH5α [[File:Psright.png|20px]]
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08/19/13
08/19/13
* Sequence analysis of BBa_K1155007 [[File:Psright.png|20px]]
* Sequence analysis of BBa_K1155007 [[File:Psright.png|20px]]
 +
 +
'''WE OBTAIN OUR BIOBRICK.'''
=='''BBa_K1155008'''==
=='''BBa_K1155008'''==
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08/01/13
08/01/13
-
* Gel purification of PCR products : FNR Part I, FNR Part II, RBS-FNR Part I PSB1C3 [[File:Psright.png|20px]]
+
* Gel purification of PCR products : FNR Part I, FNR Part II, RBS-FNR Part I pSB1C3 [[File:Psright.png|20px]]
* Gibson assembly
* Gibson assembly
Line 443: Line 445:
08/27/13
08/27/13
* Electrophoresis of FNR Part I , FNR Part II, RBS-FNR Part I [[File:Psright.png|20px]]
* Electrophoresis of FNR Part I , FNR Part II, RBS-FNR Part I [[File:Psright.png|20px]]
-
* Electrophoresis of PSB1C3 [[File:Pswrong.png|20px]]
+
* Electrophoresis of pSB1C3 [[File:Pswrong.png|20px]]
-
* Digestion of PBB1C3 by DnpI to clean it
+
* Digestion of pBB1C3 by DnpI to clean it
-
* Electrophoresis of the digestion of PSB1C3 by DnpI [[File:Psright.png|20px]]
+
* Electrophoresis of the digestion of pSB1C3 by DnpI [[File:Psright.png|20px]]
08/28/13
08/28/13
-
* Gel purification of PSB1C3 digested by DnpI
+
* Gel purification of pSB1C3 digested by DnpI
-
* Electrophoresis to check the gel purification of PSB1C3 digested by DnpI [[File:Psright.png|20px]]
+
* Electrophoresis to check the gel purification of pSB1C3 digested by DnpI [[File:Psright.png|20px]]
* Gibson assembly of RSB-FRN, FNR,
* Gibson assembly of RSB-FRN, FNR,
* Transformation of FNR, RBS-FNR in DH5α [[File:Psright.png|20px]]
* Transformation of FNR, RBS-FNR in DH5α [[File:Psright.png|20px]]
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07/09/13  
07/09/13  
* PCR purification of BphR2
* PCR purification of BphR2
-
* Digestion PCR products : BphR2 and digestion of PSB1C3
+
* Digestion PCR products : BphR2 and digestion of pSB1C3
-
* Denaturation of EcoRI/PstI used for the digestion of PCR products : BphR2 and PSB1C3
+
* Denaturation of EcoRI/PstI used for the digestion of PCR products : BphR2 and pSB1C3
-
* Ligation of BphR2 and PSB1C3
+
* Ligation of BphR2 and pSB1C3
07/10/13
07/10/13
-
* Transformation of ligation of PSB1C3 and BphR2 [[File:Psright.png|20px]]
+
* Transformation of ligation of pSB1C3 and BphR2 [[File:Psright.png|20px]]
07/11/13
07/11/13
-
* Colony PCR of BphR2 in PSB1C3 in DH5α to check good insertion of BphR2 in PSB1C3
+
* Colony PCR of BphR2 in pSB1C3 in DH5α to check good insertion of BphR2 in pSB1C3
-
* Electrophoresis of Colony PCR products : BphR2 in PSB1C3 [[File:Psright.png|20px]]
+
* Electrophoresis of Colony PCR products : BphR2 in pSB1C3 [[File:Psright.png|20px]]
07/12/13
07/12/13
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07/15/13
07/15/13
-
* Digestion of BphR2 in PSB1C3 by EcoRI/PstI and SacII to check the size of fragments
+
* Digestion of BphR2 in pSB1C3 by EcoRI/PstI and SacII to check the size of fragments
-
* PCR of digestion of BphR2 in PSB1C3
+
* PCR of digestion of BphR2 in pSB1C3
07/16/13
07/16/13
-
* Electrophoresis of the digestion of BphR2 in PSB1C3 [[File:Pswrong.png|20px]]
+
* Electrophoresis of the digestion of BphR2 in pSB1C3 [[File:Pswrong.png|20px]]
07/30/13
07/30/13
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08/01/13
08/01/13
-
* Gel purification of PCR products : BphR2 Part I, BphR2 Part II PSB1C3 [[File:Psright.png|20px]]
+
* Gel purification of PCR products : BphR2 Part I, BphR2 Part II, pSB1C3 [[File:Psright.png|20px]]
* Gibson assembly
* Gibson assembly
* PCR of : RBS-BphR2 Part I
* PCR of : RBS-BphR2 Part I
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08/27/13
08/27/13
* Electrophoresis of RBS-BphR2 Part I, BphR2 Part II [[File:Psright.png|20px]]
* Electrophoresis of RBS-BphR2 Part I, BphR2 Part II [[File:Psright.png|20px]]
-
* Electrophoresis of PSB1C3 [[File:Pswrong.png|20px]]
+
* Electrophoresis of pSB1C3 [[File:Pswrong.png|20px]]
-
* Digestion of PBB1C3 by DnpI tto clean it
+
* Digestion of pSB1C3 by DnpI tto clean it
-
* Electrophoresis of the digestion of PSB1C3 by DnpI [[File:Psright.png|20px]]
+
* Electrophoresis of the digestion of pSB1C3 by DnpI [[File:Psright.png|20px]]
08/28/13
08/28/13
-
* Gel purification of PSB1C3 digested by DnpI
+
* Gel purification of pSB1C3 digested by DnpI
-
* Electrophoresis to check the gel purification of PSB1C3 digested by DnpI [[File:Psright.png|20px]]
+
* Electrophoresis to check the gel purification of pSB1C3 digested by DnpI [[File:Psright.png|20px]]
* Gibson assembly of RBS-BphR2
* Gibson assembly of RBS-BphR2
* Transformation of RBS-BphR2 in DH5α [[File:Psright.png|20px]]
* Transformation of RBS-BphR2 in DH5α [[File:Psright.png|20px]]
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* Electrophoresis of Colony PCR of RBS-BphR2 [[File:Psright.png|20px]]
* Electrophoresis of Colony PCR of RBS-BphR2 [[File:Psright.png|20px]]
-
=='''PSB3K3'''==
+
=='''pSB3K3'''==
07/09/13
07/09/13
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07/16/13
07/16/13
-
* Digestion of PSB3K3 by EcoRI/PstI
+
* Digestion of pSB3K3 by EcoRI/PstI
-
* Electrophoresis of the digestion of PSB3K3 by EcoRI/PstI [[File:Psright.png|20px]]
+
* Electrophoresis of the digestion of pSB3K3 by EcoRI/PstI [[File:Psright.png|20px]]
-
* Gel purification of electrophoresis of the digestion of PSB3K3 by EcoRI/PstI [[File:Psright.png|20px]]
+
* Gel purification of electrophoresis of the digestion of pSB3K3 by EcoRI/PstI [[File:Psright.png|20px]]
08/06/13
08/06/13
Line 591: Line 593:
* Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion [[File:Psright.png|20px]]
* Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion [[File:Psright.png|20px]]
* Electrophoresis of BBa_J004450 digested by EcoRI/PstI [[File:Psright.png|20px]]
* Electrophoresis of BBa_J004450 digested by EcoRI/PstI [[File:Psright.png|20px]]
-
* Electroelution of PSB3K3 digested by EcoRI/PstI [[File:Pswrong.png|20px]]
+
* Electroelution of pSB3K3 digested by EcoRI/PstI [[File:Pswrong.png|20px]]
08/13/13
08/13/13
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08/07/13
08/07/13
-
* Tranduction of Km in MG1655Z1 [[File:Pswrong.png|20px]]
+
* Transduction of Km in MG1655Z1 [[File:Pswrong.png|20px]]
08/08/13
08/08/13
-
* Tranduction of Km in MG1655Z1 [[File:Psright.png|20px]]
+
* Transduction of Km in MG1655Z1 [[File:Psright.png|20px]]
08/09/13
08/09/13
-
* Tranduction of Km in MG1655Z1 (part II) [[File:Psright.png|20px]]
+
* Transduction of Km in MG1655Z1 (part II) [[File:Psright.png|20px]]
08/12/13
08/12/13
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* Digestion of BBa_K1155000 by SpeI/PstI
* Digestion of BBa_K1155000 by SpeI/PstI
* Denaturation of SpeI/PstI used for the digestion of BBa_K1155000 [[File:Psright.png|20px]]
* Denaturation of SpeI/PstI used for the digestion of BBa_K1155000 [[File:Psright.png|20px]]
-
* Ligation of Pfnr with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3
+
* Ligation of Pndh* with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
-
* Transformation of ligation of Pfnr with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3 [[File:Psright.png|20px]]
+
* Transformation of ligation of Pndh* with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3 [[File:Psright.png|20px]]
* Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20 [[File:Psright.png|20px]]
* Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20 [[File:Psright.png|20px]]
08/27/13
08/27/13
-
* Colony PCR of ligation Pfnr with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3
+
* Colony PCR of ligation Pndh* with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
-
* Electrophoresis of the colony PCR products : Pfnr with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3 [[File:Psright.png|20px]]
+
* Electrophoresis of the colony PCR products : Pndh* with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3 [[File:Psright.png|20px]]
-
* Streak of Pfnr with RBS-LacZ-Term in PSB1C3 and Pfnr with RBS-Amil CP-Term in PSB1C3 to purify them [[File:Psright.png|20px]]
+
* Streak of Pndh* with RBS-LacZ-Term in pSB1C3 and Pfnr with RBS-Amil CP-Term in pSB1C3 to purify them [[File:Psright.png|20px]]
* Culture of strain MG1655Z1 Δfnr::Km containing plasmid pcp20 [[File:Psright.png|20px]]
* Culture of strain MG1655Z1 Δfnr::Km containing plasmid pcp20 [[File:Psright.png|20px]]
08/28/13
08/28/13
-
* Purification of colony transformed with ligation : Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions [[File:Pswrong.png|20px]]
+
* Purification of colony transformed with ligation : Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions [[File:Pswrong.png|20px]]
* Culture of mutant strain MG1655Z1 Δfnr with LB [[File:Psright.png|20px]]
* Culture of mutant strain MG1655Z1 Δfnr with LB [[File:Psright.png|20px]]
08/29/13
08/29/13
-
* Purification of colony transformed with ligation : Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions [[File:Pswrong.png|20px]]
+
* Purification colony of strain MG1655Z1 Δfnr with kanamycin, chloramphenicol, ampicilin or LB
-
* Colony  PCR of Pfnr wit RBS_AmilCP-Term  in DH5α
+
* Purification of colony transformed with ligation : Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions [[File:Pswrong.png|20px]]
 +
* Colony  PCR of Pndh* wit RBS_AmilCP-Term  in DH5α
* Digestion of BBa_K1155000, BBa_K1155003, BBa_K1155007 by Xbal/PstI
* Digestion of BBa_K1155000, BBa_K1155003, BBa_K1155007 by Xbal/PstI
* Electrophoresis to check the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI [[File:Psright.png|20px]]
* Electrophoresis to check the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI [[File:Psright.png|20px]]
* Gel purification of the digestion of Bba_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI
* Gel purification of the digestion of Bba_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI
* Electrophoresis to check the gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI [[File:Psright.png|20px]]
* Electrophoresis to check the gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI [[File:Psright.png|20px]]
-
* Liquid culture in aerobic or anaerobic conditions of Pfnr with RBS-Amil CP-Term in PSB1C3 [[File:Psright.png|20px]]
+
* Liquid culture in aerobic or anaerobic conditions of Pndh* with RBS-Amil CP-Term in pSB1C3 [[File:Psright.png|20px]]
-
'''WE CHARACTERIZE BBa_K1155000'''
+
'''WE CHARACTERIZE BBa_K1155000. BBa_K1155003 WORKS TOO.'''
[[File:PsPfnr3008.jpg|600px]]
[[File:PsPfnr3008.jpg|600px]]
08/30/13
08/30/13
-
* Ligation of Pfnr with RBS-Amil CP-Term in PSB1C3
+
* Ligation of Pndh* with RBS-Amil CP-Term in pSB1C3
-
* Transformation of ligation of Pfnr with RBS-Amil CP-Term in PSB1C3 in DH5α [[File:Psright.png|20px]]
+
* Transformation of ligation of Pndh* with RBS-Amil CP-Term in pSB1C3 in DH5α [[File:Psright.png|20px]]
-
* Ligation of Pfnr with RBS-LacZ-Term in PSB3K3
+
* Ligation of Pndh* with RBS-LacZ-Term in pSB3K3
-
* Transformation of ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and in DH5α [[File:Pswrong.png|20px]]
+
* Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB3K3 and in DH5α [[File:Psright.png|20px]]
-
* Electrophoresis of Colony PCR of Pfnr with RBS_AmilCP-Term  in DH5α [[File:Psright.png|20px]]
+
* Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB3C3 and in DH5α [[File:Pswrong.png|20px]]
 +
* Electrophoresis of Colony PCR of Pndh* with RBS_AmilCP-Term  in DH5α [[File:Psright.png|20px]]
 +
* Result of the purification colony of MG1655Z1 Δfnr in ampicilin, chloramphenicol, kanamycin or LB medium [File:Psright.png|20px]]
-
=='''Characterize BBa_K1155004'''==
+
'''WE OBTAIN STRAIN  MG1655Z1 Δfnr'''
 +
 
 +
[[File:PsLB3008.jpg|311px]][[File:PsKm3008.jpg|300px]]
 +
[[File:PsCm3008.jpg|311px]][[File:PsAm3008.jpg|311px]]
 +
 
 +
=='''Characterization BBa_K1155004'''==
08/12/13
08/12/13
Line 721: Line 731:
08/19/13
08/19/13
* EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
* EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
-
* Ligation of NirB with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3
+
* Ligation of PnirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3
-
* Transformation of NirB with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α [[File:Psright.png|20px]]
+
* Transformation of PnirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α [[File:Psright.png|20px]]
08/21/13
08/21/13
-
* Colony PCR of NirB with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α
+
* Colony PCR of PnirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α
08/26/13
08/26/13
* Digestion of BBa_K1155004 by SpeI/PstI
* Digestion of BBa_K1155004 by SpeI/PstI
* Denaturation of SpeI/PstI used for the digestion of BBa_K1155004 [[File:Psright.png|20px]]
* Denaturation of SpeI/PstI used for the digestion of BBa_K1155004 [[File:Psright.png|20px]]
-
* Ligation of NirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3
+
* Ligation of PnirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
-
* Transformation of ligation of NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3 [[File:Psright.png|20px]]
+
* Transformation of ligation of PnirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3 [[File:Psright.png|20px]]
08/27/13
08/27/13
-
* Colony PCR of ligation NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3
+
* Colony PCR of ligation PnirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
-
* Electrophoresis of the colony PCR products : NirB with RBS-LacZ-Term [[File:Pswrong.png|20px]]
+
* Electrophoresis of the colony PCR products : PnirB with RBS-LacZ-Term [[File:Pswrong.png|20px]]
-
* Electrophoresis of the colony PCR products : NirB with RBS-Amil CP-Term in PSB1C3 [[File:Psright.png|20px]]
+
* Electrophoresis of the colony PCR products : PnirB with RBS-Amil CP-Term in pSB1C3 [[File:Psright.png|20px]]
-
* Streak of NirB with RBS-Amil CP-Term in PSB1C3 to purify them [[File:Psright.png|20px]]
+
* Streak of PnirB with RBS-Amil CP-Term in pSB1C3 to purify them [[File:Psright.png|20px]]
08/28/13
08/28/13
-
* Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, NirB with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions [[File:Pswrong.png|20px]]
+
* Purification of colony transformed with ligation : PnirB with RBS-LacZ-Term in pSB1C3, PnirB with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions [[File:Pswrong.png|20px]]
08/29/13
08/29/13
-
* Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3 by streaking in aerobic or anaerobic conditions [[File:Pswrong.png|20px]]
+
* Purification of colony transformed with ligation : PnirB with RBS-LacZ-Term in pSB1C3 by streaking in aerobic or anaerobic conditions [[File:Pswrong.png|20px]]
* Electrophoresis to check the digestion of BBa_K1155004 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI [[File:Pswrong.png|20px]]
* Electrophoresis to check the digestion of BBa_K1155004 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI [[File:Pswrong.png|20px]]
-
=='''Characterize BBa_K1155005'''==
+
=='''Characterization of BBa_K1155005'''==
08/12/13
08/12/13
Line 772: Line 782:
08/19/13
08/19/13
* EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
* EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
-
* Ligation of NarG with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3
+
* Ligation of PnarG with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3
-
* Transformation of NarG with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α [[File:Psright.png|20px]]
+
* Transformation of PnarG with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α [[File:Psright.png|20px]]
08/21/13
08/21/13
-
* Colony PCR of NarG with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α
+
* Colony PCR of PnarG with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α
08/26/13
08/26/13
Line 785: Line 795:
* Electrophoresis to check the digestion of BBa_K1155005 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI [[File:Pswrong.png|20px]]
* Electrophoresis to check the digestion of BBa_K1155005 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI [[File:Pswrong.png|20px]]
-
=='''Characterize BBa_K1155006'''==
+
=='''Characterization of BBa_K1155006'''==
08/12/13
08/12/13
Line 810: Line 820:
08/19/13
08/19/13
* EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
* EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
-
* Ligation of NarK with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3
+
* Ligation of PnarK with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3
-
* Transformation of NarK with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α [[File:Psright.png|20px]]
+
* Transformation of PnarK with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α [[File:Psright.png|20px]]
08/21/13
08/21/13
-
* Colony PCR of NarK with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α
+
* Colony PCR of PnarK with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α
08/26/13
08/26/13
Line 828: Line 838:
08/30/13
08/30/13
-
* Ligation of NarK with RBS-LacZ-Term in PSB3K3
+
* Ligation of PnarK with RBS-LacZ-Term in pSB3K3
-
* Transformation of ligation of NarK with RBS-LacZ-Term in PSB3K3 in DH5α [[File:Psright.png|20px]]
+
* Transformation of ligation of PnarK with RBS-LacZ-Term in pSB3K3 in DH5α [[File:Psright.png|20px]]
 +
 
 +
'''BBa_K1155006 WORKS IN ANAEROBIC CONDITIONS. BBa_K1155007 WORKS.'''
 +
 
 +
[[File:PsNarKK33108.jpg|600px]]
Written by Nadia
Written by Nadia
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 00:59, 5 October 2013

Labwork Overview Biobricks Protocols Primers Safety

Contents

Labwork overview

BBa_K1155000

Engineered promoter region of E. coli Pndh gene that allows repression of the expression of the downstream gene in anaerobic conditions

07/01/13

  • PCR of pSB1C3 and Pndh*
  • Digestion of pSB1C3 plasmid and Pndh* PCR products by EcoRI/ PstI
  • Ligation of pSB1C3 and Pndh*

07/02/13

  • Transformation of E. coli DH5α with the ligation mix Psright.png

07/03/13

  • PCR on colonies obtained after the transformation to check good insertion of Pndh* in pSB1C3
  • Colonies assayed by PCR streaked on plates and used for liquid cultures

07/04/13

  • Estimation of the expected PCR size fragments using bio-informatic tools
  • Gel Electrophoresis of PCR products obtained the day before Psright.png
  • Glycerol Stocks of the clones obtained
  • DNA Extraction of the plasmids from DH5α
  • Digestion of the plasmids by NotI, MluI, HpaI to check with a different method the good insertion of Pndh* in pSB1C3
  • New liquid culture of the correct clone

07/05/13

  • Electrophoresis of the digestion of the correct clone, now named BBa_K1155000 by NotI, HpaI, MluI Pswrong.png
  • Extraction of plasmids DNA of BBa_K1155000 from DH5α for sequencing

07/15/13

  • Sequence analysis of BBa_K1155000 Psright.png

WE OBTAINED OUR BIOBRICK.

BBa_K1155001

07/09/13

  • PCR and purification of PbphbR1 DNA fragment
  • Digestion PCR products : PbphR1 and pSB1C3 by EcoRI/PstI
  • Denaturation of EcoRI/PstI enzymes used for the digestion of PCR products : PbphR1 and pSB1C3
  • Ligation of PbphR1 and pSB1C3 fragments

07/10/13

  • Transformation of E. coli with ligation mix (pSB1C3 and PbphR1)Psright.png

07/11/13

  • PCR on colonies obtained after the transformation to check good insertion of PbphR1 in pSB1C3
  • Gel electrophoresis of PCR products : clones 5 and 6 correct Pswrong.png

07/12/13

  • Glycerol stock of clones 5, 6
  • DNA plasmid extraction of clones 5, 6

07/15/13

  • Digestion of BBa_K1155001 by EcoRI/PstI and SacII to check the size of fragments
  • PCR of digestion of BBa_K1155001

07/16/13

  • Gel electrophoresis of the digestion of BBa_K1155001 Psright.png

07/17/13

  • Glycerol Stock of BBa_K1155001
  • DNA plasmid extraction of BBa_K1155001 from DH5α

07/24/13

  • Sequence analysis of BBa_K1155001 Psright.png

WE OBTAINED OUR BIOBRICK.

BBa_K1155002

07/09/13

  • PCR and purification of PbphA1
  • Digestion PCR products : PbphA1 and pSB1C3 by EcoRI/PstI
  • Denaturation of EcoRI/PstI enzymes used for the digestion of PCR products : PbphA1 and pSB1C3
  • Ligation of PbphA1 and pSB1C3

07/10/13

  • Transformation of E. coli DH5α with the ligation mix Psright.png

07/11/13

  • PCR on colonies obtained after the transformation to check good insertion of PbphA1 in pSB1C3
  • Gel electrophoresis of PCR products : BBa_K1155002 in PSB1C3 Pswrong.png

07/12/13

  • Stock of clones 5, 6, 7, 8 for BBa_K1155002
  • Streak of BBa_K1155002
  • DNA plasmid extraction of clones 5, 6, 7, 8 for BBa_K1155002 from DH5α

07/15/13

  • Digestion of BBa_K1155002 by EcoRI/PstI and SacII to check the size of fragments
  • PCR of digestion of BBa_K1155002

07/16/13

  • Gel electrophoresis of the digestion of BBa_K1155002 Pswrong.png

07/25/13

  • New ligation of PbphA1 in pSB1C3

07/26/13

  • Transformation of E. coli DH5α with the ligation mix Psright.png

07/30/13

  • PCR on colonies obtained after the transformation to check good insertion of PbphA1 in pSB1C3
  • Gel electrophoresis of the PCR products Psright.png

08/06/13

  • Sequence analysis for BBa_K1155002 in clones 4, 17 and 22

WE OBTAIN OUR BIOBRICK.

BBa_K1155003

07/02/13

  • Tranformation of E. coli DH5α with BBa_K592009 Psright.png

07/03/13

  • New culture of BBa_K592009

07/05/13

  • Extraction of BBa_K592009 from E. coli DH5α

07/08/13

  • Digestion of BBa_K592009 by NotI, XhoI, EcoRI, EcoRI/PstI
  • Electrophoresis of the digestion of BBa_K592009 by NotI, XhoI, EcoRI, EcoRI/PstI Psright.png

07/12/13

  • Transformation of E. coli DH5α with BBa_B0015, BBa_B0017 Psright.png
  • Design of RBS-AmilCP primers

07/15/13

  • Liquid culture of BBa_B0015, BBa_B0017 Psright.png

07/16/13

  • Plasmidic DNA extraction of BBa_B0015, BBa_B0017 from E. coli DH5α
  • Digestion of BBa_B0015, BBa_B0017, BBa_I732019 by EcoRI/PstI

07/18/13

  • PCR of BBa_B0015, BBa_B0017 digested by EcoRI/PstI

07/19/13

  • Gel electrophoresis of PCR products : BBa_B0015, BBa_B0017 Psright.png
  • Gel purification of the electrophoresis of PCR products : BBa_B0015, BBa_B0017 Psright.png

07/22/13

  • Digestion of BBa_K592009 by SpeI/PstI, BBa_B0015 and BBa_B0017 by PstI/XbaI
  • Denaturation of SpeI,PstI and PstI,XbaI enzymes used for the digestion of BBa_K592009, BBa_B0015 and BBa_B0017

07/23/13

  • Gel electrophoresis of the digestion of BBa_K592009 by SpeI/PstI Pswrong.png
  • Gel electrophoresis of the digestion of BBa_B0015, BBa_B0017 by XbaI/PstI Psright.png
  • Digestion of BBa_K592009 by SpeI/PstI
  • Gel electrophoresis to check the digestion of BBa_K592009 by SpeI/PstI Pswrong.png
  • Liquid culture of BBa_K592009 in E. coli DH5α Psright.png

07/24/13

  • Plasmid extraction of BBa_K592009 from E. coli DH5α

07/25/13

  • Gel purification of the terminator
  • Gel electrophoresis to check the gel purification of Term Psright.png
  • PCR of BBa_K592209 with the primer : RBS-Amil CP and suffix_pSB1C3
  • Digestion of PCR products : RBS-Amil CP by EcoRI/SpeI

07/26/13

  • Denaturation of EcoRI/SpeI enzymes used for the digestion of PCR product : RBS-AmilCP
  • Gel Electrophoresis of the digestion of PCR products : RBS-AmilCP Psright.png
  • Ligation of RBS-AmilCP and Term-pSB1C3

07/30/13

  • Tranformation of E. coli DH5α with the ligation mix Psright.png

08/02/13

  • DNA plasmid extraction
  • Electrophoresis to check the plasmid extraction Psright.png

08/05/13

  • Digestion of BBa_K1155003 by EcoRI/PstI to check the cloning between RBS-Amil CP,Term and pSB1C3
  • Gel electrophoresis of the digestion of BBa_K1155003 Psright.png

08/07/13

  • Sequensis analysis of BBa_K1155003 [File:Psright.png|20px]]

WE OBTAIN OUR BIOBRICK.

BBa_K1155004

07/25/13

  • PCR to amplify the promoter of nirB
  • Gel Electrophoresis to check the correct amplification of PnirB Pswrong.png
  • PCR to amplify the promoter of nirB

07/26/13

  • Gel Electrophoresis to check the correct amplification of PnirB Pswrong.png
  • PCR of PnirB
  • Electrophoresis of PCR products : PnirB Psright.png
  • Gel purification of PCR products : PnirB Psright.png
  • Digestion of PCR products : PnirB by EcoRI/PstI

07/30/13

  • Denaturation of EcoRI/SpeI used of the digestion of PCR products : PnirB

07/31/13

  • Digestion of PSB1C3 by EcoRI/PstI'
  • Denaturation of EcoRI/PstI used for the digestion of PSB1C3
  • Ligation of PnirB in pSB1C3
  • Electrophoresis to check the ligation of PnirB in pSB1C3 Psright.png
  • Transformation of BBa_K1155004 Psright.png

08/05/13

  • Colony PCR of BBa_K1155004 in DH5α

08/06/13

  • Electrophoresis to check the Colony PCR products : BBa_K1155004 Psright.png
  • Liquid culture of DH5α with BBa_K1155004 Psright.png

08/20/13

  • Sequences analysis Psright.png

WE OBTAIN OUR BIOBRICK

BBa_K1155005

07/25/13

  • Colony PCR of PnarG
  • Electrophoresis to check the colony PCR of PnarG Pswrong.png
  • Colony PCR of PnarG

07/26/13

  • Electrophoresis of PCR products : PnarG Pswrong.png
  • PCR of PnarG
  • Electrophoresis of PCR products : PnarG Psright.png
  • Gel purification of PCR products : PnarG Psright.png
  • Digestion of PCR products : PnarG by EcoRI/PstI

07/30/13

  • Denaturation of EcoRI/SpeI used of the digestion of PCR products : PnarG

07/31/13

  • Digestion of pSB1C3 by EcoRI/PstI'
  • Denaturation of EcoRI/PstI used for the digestion of pSB1C3
  • Ligation of PnarG in pSB1C3
  • Electrophoresis to check the ligation of PnarG in pSB1C3 Psright.png
  • Transformation of BBa_K1155005 Psright.png

08/05/13

  • Colony PCR of BBa_K115005 in DH5α

08/06/13

  • Electrophoresis to check the Colony PCR products : BBa_K1155005 Psright.png
  • Liquid culture of DH5α with BBa_K1155005 Psright.png

08/20/13

  • Sequences analysis Psright.png

WE OBTAIN OUR BIOBRICK

BBa_K1155006

07/25/13

  • Colony PCR of PnarK
  • Electrophoresis to check the colony PCR of PnarK Pswrong.png
  • Colony PCR of PnarK

07/26/13

  • Electrophoresis of PCR products : PnarK Pswrong.png
  • PCR of PnarK
  • Electrophoresis of PCR products : PnarK Psright.png
  • Gel purification of PCR products : PnarK Psright.png
  • Digestion of PCR products : PnarK by EcoRI/PstI

07/30/13

  • Denaturation of EcoRI/SpeI used of the digestion of PCR products : PnarK

07/31/13

  • Digestion of pSB1C3 by EcoRI/PstI'
  • Denaturation of EcoRI/PstI used for the digestion of pSB1C3
  • Ligation of PnarK in pSB1C3
  • Electrophoresis to check the ligation of PnarK in pSB1C3 Psright.png
  • Transformation of BBa_K1155006 Psright.png

08/05/13

  • Colony PCR of BBa_K1155006 in DH5α

08/06/13

  • Electrophoresis to check the Colony PCR products : BBa_K1155006 Psright.png
  • Liquid culture of DH5α with BBa_K1155006 Psright.png

08/20/13

  • Sequences analysis Psright.png

WE OBTAIN OUR BIOBRICK

BBa_K1155007

07/02/13

  • Tranformation of BBa_I732017 in DH5α Psright.png

07/03/13

  • New culture of BBa_I732017

07/05/13

  • Extraction of BBa_I732017 from DH5α

07/08/13

  • Digestion of BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI
  • Electrophoresis of the digestion of BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI Psright.png

07/10/13

  • Transformation of BBa_I732019 in DH5α Pswrong.png

07/12/13

  • Transformation of BBa_I732019, BBa_B0015, BBa_B0017 Psright.png

07/15/13

  • Liquid culture of BBa_B0015, BBa_B0017, BBa_I732019 Psright.png

07/16/13

  • Extraction of BBa_B0015, BBa_B0017, BBa_I732019 from DH5α
  • Digestion of BBa_B0015, BBa_B0017, BBa_I732019 by EcoRI/PstI

07/17/13

  • Electrophoresis of digestions of BBa_B0015, BBa_B0017 by EcoRI/PstI Psright.png
  • Electrophoresis of digestions of BBa_I732019 by EcoRI/PstI Pswrong.png

07/18/13

  • PCR of BBa_B0015, BBa_B0017 digested by EcoRI/PstI

07/19/13

  • Electrophoresis of Colony PCR products : BBa_B0015, BBa_B0017 Psright.png
  • Gel purification of the electrophoresis of Colony PCR products : BBa_B0015, BBa_B0017 Psright.png

07/22/13

  • Digestion of BBa_I732017 by EcoRI/SpeI, BBa_B0015 and BBa_B0017 by PstI/XbaI
  • Denaturation of EcoRI/SpeI and PstI/XbaI used for the digestion of BBa_I732017, BBa_K502009, BBa_B0015 and BBa_B0017

07/23/13

  • Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI, BBa_B0015, BBa_B0017 by XbaI/PstI Psright.png
  • Gel purification of digestion products : RBS-LacZ
  • Electrophoresis to check the gel purification of digestion products : RBS-LacZ Pswrong.png
  • Digestion of BBa_I732017 by EcoRI/Pst
  • Electrophoresis to check the gel digestion of BBa_I732017 by EcoRI/SpeI Pswrong.png
  • Liquid culture of BBa_I732017 in DH5α Psright.png

07/24/13

  • Extraction of BBa_I732017 from DH5α

07/25/13

  • Gel purification of Term
  • Electrophoresis to check the gel purification of Term Psright.png

07/30/13

  • Digestion of BBa_I732017 by EcoRI/PstI
  • Digestion of BBa_I732017 by EcoRI/SpeI
  • Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI
  • Gel purification of digestion of BBa_I732017 by EcoRI/SpeI Pswrong.png

07/31/13

  • Digestion of BBa_I732017 by EcoRI/SpeI
  • Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI Psright.png
  • Gel purification of digestion of RBS-LacZ Pswrong.png

08/02/13

  • Digestion of BBa_I732017 by EcoRI/SpeI to check sizes of fragments
  • Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI Psright.png

08/05/13

  • Digestion of BBa_I732017 by EcoRI/SpeI
  • Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI Psright.png
  • PCR of pSB1C3
  • Electrophoresis of PCR of pSB1C3 Psright.png
  • Gel purification of electrophoresis of PCR of pSB1C3 Psright.png

08/06/13

  • Electroelution of BBa_I732017 digested by EcoRI/SpeI Psright.png

08/07/13

  • Ligation of RBS-LacZ-Term in pSB1C3
  • Tranformation BBa_K115507 in DH5α Psright.png

08/08/13

  • Colony PCR of BBa_K115007 in DH5α
  • Electrophoresis to check the colony PCR products : Bba_K1155007 Psright.png

08/09/13

  • Extraction of BBa_K115007 from DH5α

08/19/13

  • Sequence analysis of BBa_K1155007 Psright.png

WE OBTAIN OUR BIOBRICK.

BBa_K1155008

07/30/13

  • PCR of : FNR Part I, FNR Part II, RBS-FNR Part I
  • Electrophoresis of the PCR products : FNR Part I, FNR Part II, RBS-FNR Part I Psright.png

08/01/13

  • Gel purification of PCR products : FNR Part I, FNR Part II, RBS-FNR Part I pSB1C3 Psright.png
  • Gibson assembly

08/02/13

  • Tranformation of FNR and RBS-FNR in DH5α Pswrong.png

08/08/13

  • Extraction of plasmid of FNR, RBS-FNR

08/09/13

  • Electrophoresis of the PCR of FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification Pswrong.png
  • PCR of FNR Part I, FNR Part II, RBS-FNR Part I

08/12/13

  • Electrophoresis of PCR products : RBS-FNR Part I, FNR Part I and FNR Part II

08/13/13

  • PCR of FRN Part I, FNR Part II
  • Electrophoresis of PCR products : FRN Part I, FNR Part II Psright.pngFile:Psright.png|20px]]
  • Gel purification of PCR product : FNR Part I, FNR Part II, RBS-FNR Part I Psright.png

08/14/13

  • Gibson assembly of RBS-FNR, FNR

08/21/13

  • Transformation of FNR, RBS-FNR in DH5α Pswrong.png

08/26/13

  • Gibson assembly RBS-FNR, FNR
  • Transformation of FNR, RBS-FNR in DH5α Pswrong.png

08/27/13

  • Electrophoresis of FNR Part I , FNR Part II, RBS-FNR Part I Psright.png
  • Electrophoresis of pSB1C3 Pswrong.png
  • Digestion of pBB1C3 by DnpI to clean it
  • Electrophoresis of the digestion of pSB1C3 by DnpI Psright.png

08/28/13

  • Gel purification of pSB1C3 digested by DnpI
  • Electrophoresis to check the gel purification of pSB1C3 digested by DnpI Psright.png
  • Gibson assembly of RSB-FRN, FNR,
  • Transformation of FNR, RBS-FNR in DH5α Psright.png

08/29/13

  • Colony PCR of FNR, RBS-FNR and RBS-BphR2 in DH5α

08/30/13

  • Electrophoresis of Colony PCR of FNR, RBS-FNR Psright.png

BBa_K1155009

07/01/13

  • Design of oligos for amplification of BphR2 gene of Pseudomonas pseudoalcaligenes, Pseudomonas oleovorans

07/09/13

  • PCR purification of BphR2
  • Digestion PCR products : BphR2 and digestion of pSB1C3
  • Denaturation of EcoRI/PstI used for the digestion of PCR products : BphR2 and pSB1C3
  • Ligation of BphR2 and pSB1C3

07/10/13

  • Transformation of ligation of pSB1C3 and BphR2 Psright.png

07/11/13

  • Colony PCR of BphR2 in pSB1C3 in DH5α to check good insertion of BphR2 in pSB1C3
  • Electrophoresis of Colony PCR products : BphR2 in pSB1C3 Psright.png

07/12/13

  • Stock of clones 3, 4 for BphR2
  • Streak of BphR2
  • Extraction of clones 3, 4 for BphR2 from DH5α

07/15/13

  • Digestion of BphR2 in pSB1C3 by EcoRI/PstI and SacII to check the size of fragments
  • PCR of digestion of BphR2 in pSB1C3

07/16/13

  • Electrophoresis of the digestion of BphR2 in pSB1C3 Pswrong.png

07/30/13

  • PCR of : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I
  • Electrophoresis of the PCR products : BphR2 Part I, BphR2 Part II Psright.png
  • Electrophoresis of the PCR products : RBS-BphR2 Part I Pswrong.png
  • PCR of : RBS-BphR2 Part I
  • Gel purification of PCR of : RBS-BphR2 Part I Pswrong.png

08/01/13

  • Gel purification of PCR products : BphR2 Part I, BphR2 Part II, pSB1C3 Psright.png
  • Gibson assembly
  • PCR of : RBS-BphR2 Part I
  • Electrophoresis of the PCR products : RBS-BphR2 Part I Psright.png

08/02/13

  • Tranformation of BphR2 in DH5α Pswrong.png

08/08/13

  • Extraction of plasmid of BphR2
  • Gel purification of RBS-BphR2 Part I [File:Pswrong.png|20px]]

08/09/13

  • Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I to check the gel purification Pswrong.png
  • PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I

08/12/13

  • Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II

08/13/13

  • PCR of BphR2 Part I
  • Electrophoresis of PCR products : BphR2 Part I Pswrong.png
  • Electrophoresis of PCR product : BphR2 Part I to separate BphR2 from an other stripe Pswrong.png
  • PCR of BphR2 Part I
  • Gel purification of PCR product : BphR2 Part II, RBS-BphR2 Part I Psright.png

08/14/13

  • Gibson assembly of RBS-BphR2
  • Electrophoresis of PCR products : BphR2 Part I Pswrong.png
  • PCR of BphR2 Part I

08/19/13

  • Electrophoresis gel of PCR products : BphR2 Part I Psright.png
  • Gel purification of PCR products : BphR2 Part I

08/20/13

  • Electrophoresis of PCR products : BphR2 Part I Psright.png
  • Gel purification of PCR products : BphR2 Part I Pswrong.png

08/21/13

  • Transformation of RBS-BphR2 in DH5α Pswrong.png

08/22/13

  • PCR of BphR2 Part I
  • Electrophoresis of PCR product : BphR2 Part I Psright.png
  • Gel purification of PCR product : BphR2 Part I

08/26/13

  • Gibson assembly of RBS-BphR2
  • Transformation of RBS-BphR2 in DH5α Pswrong.png

08/27/13

  • Electrophoresis of RBS-BphR2 Part I, BphR2 Part II Psright.png
  • Electrophoresis of pSB1C3 Pswrong.png
  • Digestion of pSB1C3 by DnpI tto clean it
  • Electrophoresis of the digestion of pSB1C3 by DnpI Psright.png

08/28/13

  • Gel purification of pSB1C3 digested by DnpI
  • Electrophoresis to check the gel purification of pSB1C3 digested by DnpI Psright.png
  • Gibson assembly of RBS-BphR2
  • Transformation of RBS-BphR2 in DH5α Psright.png

08/29/13

  • Colony PCR of RBS-BphR2 in DH5α

08/30/13

  • Electrophoresis of Colony PCR of RBS-BphR2 Psright.png

pSB3K3

07/09/13

  • Transformation of BBa_J04450 in DH5α Psright.png

07/10/13

  • Mini and maxi preparation of the culture of BBa_J04450 Psright.png

07/11/13

  • Extraction of BBa_J04450 from DH5α
  • Digestion of BBa_J04450 to chek the size for the plasmid
  • Electrophoresis of the digestion of BBa_J04450 Pswrong.png

07/16/13

  • Digestion of pSB3K3 by EcoRI/PstI
  • Electrophoresis of the digestion of pSB3K3 by EcoRI/PstI Psright.png
  • Gel purification of electrophoresis of the digestion of pSB3K3 by EcoRI/PstI Psright.png

08/06/13

  • Extraction of BBa_J04450 from DH5α

08/08/13

  • Digestion of BBa_J04450 by EcoRI/PstI
  • Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion Psright.png
  • Electrophoresis of BBa_J004450 digested by EcoRI/PstI Psright.png
  • Electroelution of pSB3K3 digested by EcoRI/PstI Pswrong.png

08/13/13

  • Digestion of BBa_J04450 by EcoRI/PstI
  • Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI Psright.png

08/21/13

  • Electrophoresis of the digestion of BBa_J04450 by EcoRI/PstI Psright.png

08/22/13

  • Gel purification of the digestion of BBa_J04450 by EcoRI/PstI
  • Electrophoresis of gel purification of the digestion of BBa_J04450 by EcoRI/PstI
  • Ethanol precipitation of the digestion of BBa_J04450 by EcoRI/PstI Pswrong.png

08/23/13

  • Electrophoresis to check the ethanol precipitation of the digestion of BBa_J04450 by EcoRI/PstI Pswrong.png

08/29/13

  • Digestion of BBa_J04450 by EcoRI/PstI
  • Electrophoresis to check the digestion of BBa_J04450 by EcoRI/PstI Psright.png
  • Gel purification of the digestion of BBa_J04450 by PstI/SpeI Psright.png

Characterization of BBa_K1155000

08/07/13

  • Transduction of Km in MG1655Z1 Pswrong.png

08/08/13

  • Transduction of Km in MG1655Z1 Psright.png

08/09/13

  • Transduction of Km in MG1655Z1 (part II) Psright.png

08/12/13

  • Digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBaI/PstI
  • Electrophoresis to check the digestion of BBa_K1155007 and BBa_K1155003 by XBalI/PstI Psright.png
  • Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI Pswrong.png
  • Digestion of BBa_K1155000 by SpeI/PstI

08/13/13

  • Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI Pswrong.png
  • Digestion of BBa_K1155000 by SpeI and by SpeI/EcoRI
  • Electrophoresis to check the digestion of BBa_K1155000 by SpeI and EcoRI/SpeI Pswrong.png
  • Liquid culture of MG1655Z1 Δfnr::Km

08/14/13

  • Extraction of BBa_K1155000 from DH5α
  • Digestion of BBa_K1155000 by SpeI and EsoRI/SpeI
  • Electrophoresis to check the digestion of BBa_K1155000 by SpeI Pswrong.png
  • Electrophoresis to check the digestion of BBa_K1155000 by EcoRI/SpeI Psright.png
  • Gel purification of the digestion of BBa_K1155000 by EcoRI/SpeI Pswrong.png
  • Electrophoresis of the digestion of BBa_K1155003, BBa_K1155007 by XBaI/PstI
  • Gel purification of the digestion of BBa_K1155003 and Bba_K1155007 by XBaI/PstI Psright.png
  • Glycerol stock of MG1655Z1 Δfnr::Km

08/19/13

  • EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
  • Make competent cells of strain MG1655Z1 Δfnr::Km
  • Transformation of pcp20 into strain MG1655Z1 Δfnr::Km Psright.png

08/21/13

  • Culture of BBa_K1155000 Psright.png

08/22/13

  • Plasmid extraction of BBa_K1155000 from DH5α
  • Digestion of BBa_K1155000 by SpeI

08/26/13

  • Digestion of BBa_K1155000 by SpeI/PstI
  • Denaturation of SpeI/PstI used for the digestion of BBa_K1155000 Psright.png
  • Ligation of Pndh* with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
  • Transformation of ligation of Pndh* with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3 Psright.png
  • Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20 Psright.png

08/27/13

  • Colony PCR of ligation Pndh* with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
  • Electrophoresis of the colony PCR products : Pndh* with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3 Psright.png
  • Streak of Pndh* with RBS-LacZ-Term in pSB1C3 and Pfnr with RBS-Amil CP-Term in pSB1C3 to purify them Psright.png
  • Culture of strain MG1655Z1 Δfnr::Km containing plasmid pcp20 Psright.png

08/28/13

  • Purification of colony transformed with ligation : Pndh* with RBS-LacZ-Term in pSB1C3, Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions Pswrong.png
  • Culture of mutant strain MG1655Z1 Δfnr with LB Psright.png

08/29/13

  • Purification colony of strain MG1655Z1 Δfnr with kanamycin, chloramphenicol, ampicilin or LB
  • Purification of colony transformed with ligation : Pndh* with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions Pswrong.png
  • Colony PCR of Pndh* wit RBS_AmilCP-Term in DH5α
  • Digestion of BBa_K1155000, BBa_K1155003, BBa_K1155007 by Xbal/PstI
  • Electrophoresis to check the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI Psright.png
  • Gel purification of the digestion of Bba_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI
  • Electrophoresis to check the gel purification of the digestion of BBa_K1155000 by PstI/SpeI, BBa_K1155003, BBa_K1155007 by Xbal/PstI Psright.png
  • Liquid culture in aerobic or anaerobic conditions of Pndh* with RBS-Amil CP-Term in pSB1C3 Psright.png

WE CHARACTERIZE BBa_K1155000. BBa_K1155003 WORKS TOO.

PsPfnr3008.jpg

08/30/13

  • Ligation of Pndh* with RBS-Amil CP-Term in pSB1C3
  • Transformation of ligation of Pndh* with RBS-Amil CP-Term in pSB1C3 in DH5α Psright.png
  • Ligation of Pndh* with RBS-LacZ-Term in pSB3K3
  • Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB3K3 and in DH5α Psright.png
  • Transformation of ligation of Pndh* with RBS-LacZ-Term in pSB3C3 and in DH5α Pswrong.png
  • Electrophoresis of Colony PCR of Pndh* with RBS_AmilCP-Term in DH5α Psright.png
  • Result of the purification colony of MG1655Z1 Δfnr in ampicilin, chloramphenicol, kanamycin or LB medium [File:Psright.png|20px]]

WE OBTAIN STRAIN MG1655Z1 Δfnr

PsLB3008.jpgPsKm3008.jpg PsCm3008.jpgPsAm3008.jpg

Characterization BBa_K1155004

08/12/13

  • Digestion of BBa_K1155007 and BBa_K1155003 by XBaI/PstI
  • Electrophoresis to check the digestion of BBa_K1155007 and BBa_K1155003 by XBalI/PstI Psright.png

08/13/13

  • Extraction of BBa_K1155004 from DH5α
  • Digestion of BBa_K1155004 by SpeI and by SpeI/EcoRI
  • Electrophoresis to check the digestion of BBa_K1155004 by SpeI Psright.png
  • Electrophoresis to check the digestion of BBa_K1155004 by SpeI/EcoRI Pswrong.png

08/14/13

  • Gel purification of the digestion of BBa_K1155004 by SpeI Pswrong.png
  • Extraction of BBa_K1155004 from DH5α
  • Digestion of BBa_K1155004 by EcoRI/SpeI
  • Electrophoresis to check the digestion of BBa_K1155004 by EcoRI/SpeI Pswrong.png
  • Digestion of BBa_K1155004 by SpeI and EcoRI/SpeI
  • Electrophoresis to check the digestion of BBa_K1155004 by SpeI Psright.png
  • Electrophoresis to check the digestion of BBa_K1155004 by EcoRI/SpeI Pswrong.png
  • Gel purification of the digestion of BBa_K1155004 by SpeI Psright.png
  • Electrophoresis of the digestion of BBa_K1155003, BBa_K1155007 by XBaI/PstI
  • Gel purification of the digestion of BBa_K1155003 and BBa_K1155007 by XBaI/PstI Psright.png

08/19/13

  • EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
  • Ligation of PnirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3
  • Transformation of PnirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α Psright.png

08/21/13

  • Colony PCR of PnirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α

08/26/13

  • Digestion of BBa_K1155004 by SpeI/PstI
  • Denaturation of SpeI/PstI used for the digestion of BBa_K1155004 Psright.png
  • Ligation of PnirB clone 6 with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
  • Transformation of ligation of PnirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3 Psright.png

08/27/13

  • Colony PCR of ligation PnirB with RBS-LacZ-Term or RBS-Amil CP-Term in pSB1C3
  • Electrophoresis of the colony PCR products : PnirB with RBS-LacZ-Term Pswrong.png
  • Electrophoresis of the colony PCR products : PnirB with RBS-Amil CP-Term in pSB1C3 Psright.png
  • Streak of PnirB with RBS-Amil CP-Term in pSB1C3 to purify them Psright.png

08/28/13

  • Purification of colony transformed with ligation : PnirB with RBS-LacZ-Term in pSB1C3, PnirB with RBS-Amil CP-Term in pSB1C3 by streaking in aerobic or anaerobic conditions Pswrong.png

08/29/13

  • Purification of colony transformed with ligation : PnirB with RBS-LacZ-Term in pSB1C3 by streaking in aerobic or anaerobic conditions Pswrong.png
  • Electrophoresis to check the digestion of BBa_K1155004 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI Pswrong.png

Characterization of BBa_K1155005

08/12/13

  • Digestion of BBa_K1155007 and BBa_K1155003 by XBaI/PstI
  • Electrophoresis to check the digestion of BBa_K1155007 and BBa_K1155003 by XBalI/PstI Psright.png

08/13/13

  • Extraction of BBa_K1155005 from DH5α
  • Digestion of BBa_K1155005 by SpeI and by SpeI/EcoRI
  • Electrophoresis to check the digestion of BBa_K1155005 by SpeI Psright.png
  • Electrophoresis to check the digestion of BBa_K1155005 by SpeI/EcoRI Pswrong.png

08/14/13

  • Gel purification of the digestion of BBa_K1155005 by SpeI Pswrong.png
  • Extraction of BBa_K1155005 from DH5α
  • Digestion of BBa_K1155005 by EcoRI/SpeI
  • Electrophoresis to check the digestion of BBa_K1155005 by EcoRI/SpeI Pswrong.png
  • Digestion of BBa_K1155005 by SpeI and EcoRI/SpeI
  • Electrophoresis to check the digestion of BBa_K1155005 by SpeI Psright.png
  • Electrophoresis to check the digestion of BBa_K1155005 by EcoRI/SpeI Pswrong.png
  • Gel purification of the digestion of BBa_K1155005 by SpeI Psright.png
  • Electrophoresis of the digestion of BBa_K1155003, BBa_K1155007 by XBaI/PstI
  • Gel purification of the digestion of BBa_K1155003 and BBa_K1155007 by XBaI/PstI Psright.png

08/19/13

  • EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
  • Ligation of PnarG with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3
  • Transformation of PnarG with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α Psright.png

08/21/13

  • Colony PCR of PnarG with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α

08/26/13

  • Digestion of BBa_K1155005 by SpeI/PstI
  • Denaturation of SpeI/PstI used for the digestion of BBa_K1155005 Pswrong.png

08/29/13

  • Electrophoresis to check the digestion of BBa_K1155005 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI Pswrong.png

Characterization of BBa_K1155006

08/12/13

  • Digestion of BBa_K1155007 and BBa_K1155003 by XBaI/PstI
  • Electrophoresis to check the digestion of BBa_K1155007 and BBa_K1155003 by XBalI/PstI Psright.png

08/13/13

  • Extraction of BBa_K1155006 from DH5α
  • Digestion of BBa_K1155006 by SpeI and by SpeI/EcoRI
  • Electrophoresis to check the digestion of BBa_K1155006 by SpeI Psright.png
  • Electrophoresis to check the digestion of BBa_K1155006 by SpeI/EcoRI Pswrong.png

08/14/13

  • Gel purification of the digestion of BBa_K1155006 by SpeI Pswrong.png
  • Extraction of BBa_K1155006 from DH5α
  • Digestion of BBa_K1155006 by EcoRI/SpeI
  • Electrophoresis to check the digestion of BBa_K1155006 by EcoRI/SpeI Pswrong.png
  • Digestion of BBa_K1155006 by SpeI and EcoRI/SpeI
  • Electrophoresis to check the digestion of BBa_K1155006 by SpeI and EcoRI/SpeIPsright.png
  • Gel purification of the digestion of BBa_K1155006 by EcoRI/SpeI and SpeI Psright.png
  • Electrophoresis of the digestion of BBa_K1155003, BBa_K1155007 by XBaI/PstI
  • Gel purification of the digestion of BBa_K1155003 and BBa_K1155007 by XBaI/PstI Psright.png

08/19/13

  • EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term
  • Ligation of PnarK with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3
  • Transformation of PnarK with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α Psright.png

08/21/13

  • Colony PCR of PnarK with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α

08/26/13

  • Digestion of BBa_K1155006 by SpeI/PstI
  • Denaturation of SpeI/PstI used for the digestion of BBa_K1155006 Pswrong.png

08/29/13

  • Electrophoresis to check the digestion of BBa_K1155006 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI Psright.png
  • Digestion of BBa_K1155003, BBa_K1155007 by Xbal/PstI
  • Electrophoresis to check the digestion of BBa_K1155003, BBa_K1155007 by Xbal/PstI Psright.png
  • Gel purification of the digestion of BBa_K1155003, BBa_K1155007 by Xbal/PstI
  • Electrophoresis to check the gel purification of the digestion of BBa_K1155003, BBa_K1155007 by Xbal/PstI Psright.png

08/30/13

  • Ligation of PnarK with RBS-LacZ-Term in pSB3K3
  • Transformation of ligation of PnarK with RBS-LacZ-Term in pSB3K3 in DH5α Psright.png

BBa_K1155006 WORKS IN ANAEROBIC CONDITIONS. BBa_K1155007 WORKS.

PsNarKK33108.jpg

Written by Nadia

Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Labwork"