Contents 1 Labwork overview 1.1 BBa_K1155000 1.2 BBa_K1155001 1.3 BBa_K1155002 1.4 BBa_K1155003 1.5 BBa_K1155004 1.6 BBa_K1155005 1.7 BBa_K1155006 1.8 BBa_K1155007 1.9 BBa_K1155008 1.10 BBa_K1155009 1.11 PSB3K3 1.12 Characterization of BBa_K1155000 1.13 Characterize BBa_K1155004 1.14 Characterize BBa_K1155005 1.15 Characterize BBa_K1155006

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

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
• 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
• Extraction of plasmids DNA of BBa_K1155000 from DH5α for sequencing

07/15/13

• Sequence analysis of BBa_K1155000

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)

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

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

07/17/13

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

07/24/13

• Sequence analysis of BBa_K1155001

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

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

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

07/25/13

• New ligation of PbphA1 in pSB1C3

07/26/13

• Transformation of E. coli DH5α with the ligation mix

07/30/13

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

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

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

07/12/13

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

07/15/13

• Liquid culture of BBa_B0015, BBa_B0017

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

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

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
• 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
• Ligation of RBS-AmilCP and Term-pSB1C3

07/30/13

• Tranformation of E. coli DH5α with the ligation mix

08/02/13

• DNA plasmid extraction
• Electrophoresis to check the plasmid extraction

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

BBa_K1155004

07/25/13

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

07/26/13

• Gel Electrophoresis to check the correct amplification of PnirB
• PCR of NirB
• Electrophoresis of PCR products : NirB
• Gel purification of PCR products : NarK
• Digestion of PCR products : NarK by EcoRI/PstI

07/30/13

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

07/31/13

• Digestion of PSB1C3 by EcoRI/PstI'
• Denaturation of EcoRI/PstI used for the digestion of PSB1C3
• Ligation of NirB in PSB1C3
• Electrophoresis to check the ligation of NirB in PSB1C3
• Transformation of BBa_K1155004

08/05/13

• Colony PCR of BBa_K1155004 in DH5α

08/06/13

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

08/20/13

• Sequences analysis

WE OBTAIN OUR BIOBRICK

BBa_K1155005

07/25/13

• Colony PCR of nag
• Electrophoresis to check the colony PCR of NarG
• Colony PCR of NarG

07/26/13

• Electrophoresis of PCR products : NarG
• PCR of NarG
• Electrophoresis of PCR products : NarG
• Gel purification of PCR products : NarG
• Digestion of PCR products : NarG by EcoRI/PstI

07/30/13

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

07/31/13

• Digestion of PSB1C3 by EcoRI/PstI'
• Denaturation of EcoRI/PstI used for the digestion of PSB1C3
• Ligation of NarG in PSB1C3
• Electrophoresis to check the ligation of NarG in PSB1C3
• Transformation of BBa_K1155005

08/05/13

• Colony PCR of BBa_K115005 in DH5α

08/06/13

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

08/20/13

• Sequences analysis

WE OBTAIN OUR BIOBRICK

BBa_K1155006

07/25/13

• Colony PCR of NarK
• Electrophoresis to check the colony PCR of NarK
• Colony PCR of NarK

07/26/13

• Electrophoresis of PCR products : NarK
• PCR of NarK
• Electrophoresis of PCR products : NarK
• Gel purification of PCR products : NirB
• Digestion of PCR products : NirB by EcoRI/PstI

07/30/13

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

07/31/13

• Digestion of PSB1C3 by EcoRI/PstI'
• Denaturation of EcoRI/PstI used for the digestion of PSB1C3
• Ligation of NarK in PSB1C3
• Electrophoresis to check the ligation of NarK in PSB1C3
• Transformation of BBa_K1155006

08/05/13

• Colony PCR of BBa_K1155006 in DH5α

08/06/13

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

08/20/13

• Sequences analysis

WE OBTAIN OUR BIOBRICK

BBa_K1155007

07/02/13

• Tranformation of BBa_I732017 in DH5α

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

07/10/13

• Transformation of BBa_I732019 in DH5α

07/12/13

• Transformation of BBa_I732019, BBa_B0015, BBa_B0017

07/15/13

• Liquid culture of BBa_B0015, BBa_B0017, BBa_I732019

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
• Electrophoresis of digestions of BBa_I732019 by EcoRI/PstI

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
• Gel purification of the electrophoresis of Colony PCR products : BBa_B0015, BBa_B0017

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
• Gel purification of digestion products : RBS-LacZ
• Electrophoresis to check the gel purification of digestion products : RBS-LacZ
• Digestion of BBa_I732017 by EcoRI/Pst
• Electrophoresis to check the gel digestion of BBa_I732017 by EcoRI/SpeI
• Liquid culture of BBa_I732017 in DH5α

07/24/13

• Extraction of BBa_I732017 from DH5α

07/25/13

• Gel purification of Term
• Electrophoresis to check the gel purification of Term

07/30/13

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

07/31/13

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

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

08/05/13

• Digestion of BBa_I732017 by EcoRI/SpeI
• Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI
• PCR of PSB1C3
• Electrophoresis of PCR of PSB1C3
• Gel purification of electrophoresis of PCR of PSB1C3

08/06/13

• Electroelution of BBa_I732017 digested by EcoRI/SpeI

08/07/13

• Ligation of RBS-LacZ-Term in PSB1C3
• Tranformation BBa_K115507 in DH5α

08/08/13

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

08/09/13

• Extraction of BBa_K115007 from DH5α

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

08/01/13

• Gel purification of PCR products : FNR Part I, FNR Part II, RBS-FNR Part I PSB1C3
• Gibson assembly

08/02/13

• Tranformation of FNR and RBS-FNR in DH5α

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

08/14/13

• Gibson assembly of RBS-FNR, FNR

08/21/13

• Transformation of FNR, RBS-FNR in DH5α

08/26/13

• Gibson assembly RBS-FNR, FNR
• Transformation of FNR, RBS-FNR in DH5α

08/27/13

• Electrophoresis of FNR Part I , FNR Part II, RBS-FNR Part I
• Electrophoresis of PSB1C3
• Digestion of PBB1C3 by DnpI to clean it
• Electrophoresis of the digestion of PSB1C3 by DnpI

08/28/13

• Gel purification of PSB1C3 digested by DnpI
• Electrophoresis to check the gel purification of PSB1C3 digested by DnpI
• Gibson assembly of RSB-FRN, FNR,
• Transformation of FNR, RBS-FNR in DH5α

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

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

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

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

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
• Electrophoresis of the PCR products : RBS-BphR2 Part I
• PCR of : RBS-BphR2 Part I
• Gel purification of PCR of : RBS-BphR2 Part I

08/01/13

• Gel purification of PCR products : BphR2 Part I, BphR2 Part II PSB1C3
• Gibson assembly
• PCR of : RBS-BphR2 Part I
• Electrophoresis of the PCR products : RBS-BphR2 Part I

08/02/13

• Tranformation of BphR2 in DH5α

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

08/14/13

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

08/19/13

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

08/20/13

• Electrophoresis of PCR products : BphR2 Part I
• Gel purification of PCR products : BphR2 Part I

08/21/13

• Transformation of RBS-BphR2 in DH5α

08/22/13

• PCR of BphR2 Part I
• Electrophoresis of PCR product : BphR2 Part I
• Gel purification of PCR product : BphR2 Part I

08/26/13

• Gibson assembly of RBS-BphR2
• Transformation of RBS-BphR2 in DH5α

08/27/13

• Electrophoresis of RBS-BphR2 Part I, BphR2 Part II
• Electrophoresis of PSB1C3
• Digestion of PBB1C3 by DnpI tto clean it
• Electrophoresis of the digestion of PSB1C3 by DnpI

08/28/13

• Gel purification of PSB1C3 digested by DnpI
• Electrophoresis to check the gel purification of PSB1C3 digested by DnpI
• Gibson assembly of RBS-BphR2
• Transformation of RBS-BphR2 in DH5α

08/29/13

• Colony PCR of RBS-BphR2 in DH5α

08/30/13

• Electrophoresis of Colony PCR of RBS-BphR2

PSB3K3

07/09/13

• Transformation of BBa_J04450 in DH5α

07/10/13

• Mini and maxi preparation of the culture of BBa_J04450

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

07/16/13

• Digestion of PSB3K3 by EcoRI/PstI
• Electrophoresis of the digestion of PSB3K3 by EcoRI/PstI
• Gel purification of electrophoresis of the digestion of PSB3K3 by EcoRI/PstI

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
• Electrophoresis of BBa_J004450 digested by EcoRI/PstI
• Electroelution of PSB3K3 digested by EcoRI/PstI

08/13/13

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

08/21/13

• Electrophoresis of the digestion of BBa_J04450 by EcoRI/PstI

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

08/23/13

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

08/29/13

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

Characterization of BBa_K1155000

08/07/13

• Tranduction of Km in MG1655Z1

08/08/13

• Tranduction of Km in MG1655Z1

08/09/13

• Tranduction of Km in MG1655Z1 (part II)

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
• Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI
• Digestion of BBa_K1155000 by SpeI/PstI

08/13/13

• Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI
• Digestion of BBa_K1155000 by SpeI and by SpeI/EcoRI
• Electrophoresis to check the digestion of BBa_K1155000 by SpeI and EcoRI/SpeI
• 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
• Electrophoresis to check the digestion of BBa_K1155000 by EcoRI/SpeI
• Gel purification of the digestion of BBa_K1155000 by EcoRI/SpeI
• 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
• 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

08/21/13

• Culture of BBa_K1155000

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
• Ligation of Pfnr 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
• Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20

08/27/13

• Colony PCR of ligation Pfnr 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
• Streak of Pfnr with RBS-LacZ-Term in PSB1C3 and Pfnr with RBS-Amil CP-Term in PSB1C3 to purify them
• Culture of strain MG1655Z1 Δfnr::Km containing plasmid pcp20

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

08/29/13

• Purification of colony transformed with ligation : Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions
• Colony PCR of Pfnr 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
• 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
• Liquid culture in aerobic or anaerobic conditions of Pfnr with RBS-Amil CP-Term in PSB1C3

WE CHARACTERIZE BBa_K1155000

08/30/13

• Ligation of Pfnr with RBS-Amil CP-Term in PSB1C3
• Transformation of ligation of Pfnr with RBS-Amil CP-Term in PSB1C3 in DH5α
• Ligation of Pfnr with RBS-LacZ-Term in PSB3K3
• Transformation of ligation of Pfnr with RBS-LacZ-Term in PSB3K3 and in DH5α
• Electrophoresis of Colony PCR of Pfnr with RBS_AmilCP-Term in DH5α

Characterize BBa_K1155004

08/07/13

• Tranduction of Km in MG1655Z1

08/08/13

• Tranduction of Km in MG1655Z1

08/09/13

• Tranduction of Km in MG1655Z1 (part II)

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

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
• Electrophoresis to check the digestion of BBa_K1155004 by SpeI/EcoRI
• Liquid culture of MG1655Z1 Δfnr::Km

08/14/13

• Gel purification of the digestion of BBa_K1155004 by SpeI
• Extraction of BBa_K1155004 from DH5α
• Digestion of BBa_K1155004 by EcoRI/SpeI
• Electrophoresis to check the digestion of BBa_K1155004 by EcoRI/SpeI
• Digestion of BBa_K1155004 by SpeI and EcoRI/SpeI
• Electrophoresis to check the digestion of BBa_K1155004 by SpeI
• Electrophoresis to check the digestion of BBa_K1155004 by EcoRI/SpeI
• Gel purification of the digestion of BBa_K1155004 by SpeI
• 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
• Glycerol stock of MG1655Z1 Δfnr::Km

08/19/13

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

08/21/13

• Colony PCR of NirB 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
• Ligation of NirB 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
• Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20

08/27/13

• Colony PCR of ligation NirB with RBS-LacZ-Term or RBS-Amil CP-Term in PSB1C3
• Electrophoresis of the colony PCR products : NirB with RBS-LacZ-Term
• Electrophoresis of the colony PCR products : NirB with RBS-Amil CP-Term in PSB1C3
• Streak of NirB with RBS-Amil CP-Term in PSB1C3 to purify them

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

08/29/13

• Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3 by streaking in aerobic or anaerobic conditions
• Electrophoresis to check the digestion of BBa_K1155004 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI

Characterize BBa_K1155005

08/07/13

• Tranduction of Km in MG1655Z1

08/08/13

• Tranduction of Km in MG1655Z1

08/09/13

• Tranduction of Km in MG1655Z1 (part II)

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

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
• Electrophoresis to check the digestion of BBa_K1155005 by SpeI/EcoRI
• Liquid culture of MG1655Z1 Δfnr::Km

08/14/13

• Gel purification of the digestion of BBa_K1155005 by SpeI
• Extraction of BBa_K1155005 from DH5α
• Digestion of BBa_K1155005 by EcoRI/SpeI
• Electrophoresis to check the digestion of BBa_K1155005 by EcoRI/SpeI
• Digestion of BBa_K1155005 by SpeI and EcoRI/SpeI
• Electrophoresis to check the digestion of BBa_K1155005 by SpeI
• Electrophoresis to check the digestion of BBa_K1155005 by EcoRI/SpeI
• Gel purification of the digestion of BBa_K1155005 by SpeI
• 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
• Glycerol stock of MG1655Z1 Δfnr::Km

08/19/13

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

08/21/13

• Colony PCR of NarG 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
• Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20

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

Characterize BBa_K1155006

08/07/13

• Tranduction of Km in MG1655Z1

08/08/13

• Tranduction of Km in MG1655Z1

08/09/13

• Tranduction of Km in MG1655Z1 (part II)

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

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
• Electrophoresis to check the digestion of BBa_K1155006 by SpeI/EcoRI
• Liquid culture of MG1655Z1 Δfnr::Km

08/14/13

• Gel purification of the digestion of BBa_K1155006 by SpeI
• Extraction of BBa_K1155006 from DH5α
• Digestion of BBa_K1155006 by EcoRI/SpeI
• Electrophoresis to check the digestion of BBa_K1155006 by EcoRI/SpeI
• Digestion of BBa_K1155006 by SpeI and EcoRI/SpeI
• Electrophoresis to check the digestion of BBa_K1155006 by SpeI and EcoRI/SpeI
• Gel purification of the digestion of BBa_K1155006 by EcoRI/SpeI and SpeI
• 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
• Glycerol stock of MG1655Z1 Δfnr::Km

08/19/13

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

08/21/13

• Colony PCR of NarK 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
• Purification colony from strain MG1655Z1 Δfnr::Km containing plasmid pcp20

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
• Digestion of BBa_K1155003, BBa_K1155007 by Xbal/PstI
• Electrophoresis to check the digestion of BBa_K1155003, BBa_K1155007 by Xbal/PstI
• 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

08/30/13

• Ligation of NarK with RBS-LacZ-Term in PSB3K3
• Transformation of ligation of NarK with RBS-LacZ-Term in PSB3K3 in DH5α

Written by Nadia