Revision as of 01:05, 22 September 2013

Notebook : August 30

check the size by Gel electrophoresis for PCR clonies on Gibson assembly and ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3 .
Do ligation for For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 , promoter fnr(activator)nirK + RBS_LacZ+Term_PSB3K3 and promoter fnr(repressor) + RBS_LacZ+Term_PSB3K3 and Transformation and

incubation.

[[]]

Well 1 : 6µL DNA Ladder
Well AA 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3
Well AB 1-8 : Clonies from Gibson FNR_part1, FNR part1 and plasmid PSB1C3 Concentrate3
Well AC 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
Well AD 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3
Well AE 1-8 :Clonies from Gibson RBS_FNR part1, FNR_part2 and plasmid PSB1C3 Concentrate
Well AF 1-8 :Clonies from Gibson RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3 Concentrate

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Ligation

For promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 :

Transformation theligation and spread in LB plates with Chlorenphenicol and Xgal. incubated at 37°C in aerobic condition over night.

For promoter fnr(activator)nirK + RBS_LacZ+Term_PSB3K3 :

Transformation theligation and spread in LB plates with k and Xgal. incubated at 37°C in anaerobic condition over night.

For promoter fnr(repressor) + RBS_LacZ+Term_PSB3K3 :

Transformation theligation and spread in LB plates with k and Xgal. incubated at 37°C in aerobic condition over night.

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@@ Line 157: / Line 157: @@
 =='''Lab work'''==
-==='''A - Aerobic/Anaerobic regulation system'''===
+==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
-===='''Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3'''====
+===='''Objective : obtaining FNR and BphR2 proteins'''====
-(((((((((((((((===='''1 - Electrophoresis to check the digestion of Bba_J04450 by EcoRI/PstI'''====
+===='''1 - Electrophoresis of Colony PCR of FNR, RBS-FNR and RBS-BphR2'''====
 {|
@@ Line 167: / Line 167: @@
 | style="width:350px;border:1px solid black;vertical-align:top;" |
 * Well 1 : 6µL DNA Ladder
-* Well 2 : 5µL of Bba_J04450 digested by EcoRI/PstI+1µl of 6X loading dye
+* Well 2 to 17 : 5µL of FNR+1µl of 6X loading dye
+* Well 18 to 26 : 5µL of RBS-BphR2+1µl of 6X loading dye
+* Well 27 : 6µL DNA Ladder
+* Well 28 to 41 : 5µL of RBS-FNR+1µl of 6X loading dye
+* Well 42 to 49 : 5µL of RBS-BphR2+1µl of 6X loading dye
+* WEll 50 : 6µL DNA Ladder
 * Gel : 1%
 |}
 Expect sizes :
-* PSB3K3 : 2750 bp
+* FNR : 1096 bp
+* RBS-FNR : 1014 bp
+* RBS-BphR2 : 1469 bp
 {|
 | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-We can see anything. The digestion did work.
+We obtain fragment at right size for FNR and RBS-FNR. The Gisbon assembly was good.
-|}))))))))))))))))))))))
+|}
 {{Team:Paris_Saclay/incl_fin}}