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 Characterize 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 ndh gene that allows repression of the expression of the downstream gene in anaerobic conditions

07/01/13

• 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 solution

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 plasmidic 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 purification of BphR1
• Digestion PCR products : BphR1 and digestion of PSB1C3
• Denaturation of EcoRI/PstI used for the digestion of PCR products : BphR1 and PSB1C3
• Ligation of BphR1 and PSB1C3

07/10/13

• Transformation of ligation of PSB1C3 and BphR1

07/11/13

• Colony PCR of Bba_K1155001 in DH5α to check good insertion of BphR1 in PSB1C3
• Electrophoresis of Colony PCR products : Bba_K1155001 in PSB1C3

07/12/13

• Stock of clones 5, 6 for Bba_K1155001
• Extraction of clones 5, 6 for Bba_K1155001 from DH5α

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

• Electrophoresis of the digestion of Bba_K1155001

07/17/13

• Stock of Bba_K1155001
• Extraction of Bba_K1155001 from DH5α

07/24/13

• Sequence analysis of Bba_K1155001

WE OBTAINED OUR BIOBRICK.

Bba_K1155002

07/09/13

• PCR purification of BphA1
• Digestion PCR products : BphA1 and digestion of PSB1C3
• Denaturation of EcoRI/PstI used for the digestion of PCR products : BphA1 and PSB1C3
• Ligation of BphA1 and PSB1C3

07/10/13

• Transformation of ligation of PSB1C3 and BphA1

07/11/13

• Colony PCR of Bba_K1155002 in DH5α to check good insertion of BphA1 in PSB1C3
• Electrophoresis of Colony PCR products : Bba_K1155002 in PSB1C3

07/12/13

• Stock of clones 5, 6, 7, 8 for Bba_K1155002
• Streak of Bba_K1155002
• 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

• Electrophoresis of the digestion of Bba_K1155002

07/25/13

• Ligation of BphA1 in PSB1C3

07/26/13

• Tranformation of Bba_K1155002 in DH5α

07/30/13

• Colony PCR of Bba_K1155002 in DH5α
• Electrophoresis of the Colony PCR of Bba_K1155002 in DH5α

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 Bba_K592009 in DH5α

07/03/13

• New culture of Bba_K592009

07/05/13

• Extraction of Bba_K592009 from 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 Bba_B0015, Bba_B0017
• Design of RBS-AmilCP oligos

07/15/13

• Liquid culture of Bba_B0015, Bba_B0017

07/16/13

• Extraction of Bba_B0015, Bba_B0017 from 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

• 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_K592009 by SpeI/PstI, Bba_B0015 and Bba_B0017 by PstI/XbaI
• Denaturation of SpeI/PstI and PstI/XbaI used for the digestion of Bba_K592009, Bba_B0015 and Bba_B0017

07/23/13

• Electrophoresis of the digestion of Bba_K592009 by SpeI/PstI
• Electrophoresis of the digestion of Bba_B0015, Bba_B0017 by XbaI/PstI
• Digestion of Bba_K592009 by SpeI/PstI
• Electrophoresis to check the digestion of Bba_K592009 by SpeI/PstI
• Liquid culture of Bba_K592009 in DH5α

07/24/13

• Extraction of Bba_K592009 from DH5α

07/25/13

• Gel purification of Term
• Electrophoresis to check the gel purification of Term
• PCR of Bba_K592209 with RBS-Amil CP primer and suffix : PSB1C3
• Digestion of Colony PCR products : RBS-Amil CP by EcoRI/SpeI

07/26/13

• Denaturation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP
• Ligation of RBS-AmilCP and Term-PSB1C3

07/30/13

• Tranformation of Bba_K1155003 in DH5α

08/02/13

• Extraction of Bba_K1155003 from DH5α
• Electrophoresis to check the extraction of Bba_K1155003

08/05/13

• Digestion of Bba_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and PSB1C3
• Electrophoresis of the digestion of Bba_K1155003 [File:Psright.png|20px]]

Bba_K1155004

07/25/13

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

07/26/13

• Electrophoresis of PCR products : NirB
• 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
• Culture of DH5α with Bba_K1155004

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
• Culture of DH5α with Bba_K1155005

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
• Culture of DH5α with Bba_K1155006

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

• Tranformation Bba_K115507 in 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

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

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

Characterize Bba_K1155000

Characterize Bba_K1155004

Characterize Bba_K1155005

Characterize Bba_K1155006