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1 Notebook : August 12
- 1.1 Lab work
  - 1.1.1 A - Aerobic/Anaerobic regulation system
  - 1.1.2 A - Aerobic/Anaerobic regulation system / B - PCB sensor system
    - 1.1.2.1 Objective : obtaining FNR and BphR2 proteins
    - 1.1.2.2 1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II

Notebook : August 12

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000

1 - Digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBaI/PstI

Anaïs, Nadia, XiaoJing

Used quantities :

BBa_K1155000 :
- Buffer FD : 5µL
- H2O : 38µL
- DNA : 5µL
- SpeI FD : 1µL
- PstI FD : 1µL

BBa_K1155007 :
- Buffer FD : 5µL
- H2O : 23µL
- DNA : 20µL
- XBal FD : 1µL
- PstI FD : 1µL

BBa_K1155003 :
- Buffer FD : 5µL
- H2O : 33µL
- DNA : 10µL
- XBal FD : 1µL
- PstI FD : 1µL

We incubated the digestion at 37°C during 15 minutes.

2 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBalI/PstI

Nadia

Well 1 : 6µL DNA Ladder
Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye
Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye
Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye
Gel : 0.8%

Expected sizes :

Pndh* : 111bp
RBS_LacZ_Term : 3500 kb
RBS_AmilCP_Term : 824 bp
pSB1C3 : 2070bp

We can't see any band for BBa_K1155000 digestion. The digestion failed. We will do it again. We obtain RBS-LacZ-Term and RBS-AmilCP-Term fragments. The digestion was good. We will purify it.

3 - Digestion of BBa_K1155000 by Spe I/Pst I

Anaïs, Nadia

Used quantities :

Buffer FD : 5µL
H2O : 38µL
DNA : 5µL
SpeI FD : 1µL
PstI FD : 1µL

We incubate the digestion at 37°C during 15 minutes.

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FNR and BphR2 proteins

1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II

Damir

Well 1 : 6µL DNA Ladder
Well 2 : 5µL RBS-BphR2 Part I +1µl of 6X loading dye
Well 3 : 5µL BphR2 Part II +1µl of 6X loading dye
Well 4 : 5µL FNR Part I +1µl of 6X loading dye
Well 5 : 5µL FNR Part II +1µl of 6X loading dye
Well 6 : 5µL RBS-FNR Part I +1µl of 6X loading dye
Well 7 : 5µL BphR2 Part I +1µl of 6X loading dye
Gel : 0.8%

Expected size

RBS-BphR2 Part I : 197 kb
BphR2 Part II : 790 kb
FNR Part I : 597 kb
FNR Part II : 200 kb
RBS-FNR Part I : 615 kb
BphR2 Part I : 178 kb

We can't see FNR Part I, FNR Part II and BphR2 Part I fragments at the good size. We will make the PCR again. We obtain RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.

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@@ Line 1: / Line 1: @@
 {{Team:Paris_Saclay/incl_debut_generique}}
-='''Notebook : August 9'''=
+='''Notebook : August 12'''=
 =='''Lab work'''==
@@ Line 7: / Line 7: @@
 ==='''A - Aerobic/Anaerobic regulation system'''===
-===='''1 - Obtaining Pfnr_RBS-LacZ-Term in PSB1C3'''====
+===='''Objective : characterize BBa_K1155000'''====
+===='''1 - Digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBaI/PstI'''====
 Anaïs, Nadia, XiaoJing
-We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain  was too old ( 2001)
+Used quantities :
-Protocol : [[Team:Paris_Saclay/Protocols/transduction|transduction]]
+* BBa_K1155000 :
+** Buffer FD : 5µL
+** H2O : 38µL
+** DNA : 5µL
+** SpeI FD : 1µL
+** PstI FD : 1µL
+* BBa_K1155007 :
+** Buffer FD : 5µL
+** H2O : 23µL
+** DNA : 20µL
+** XBal FD : 1µL
+** PstI FD : 1µL
+* BBa_K1155003 :
+** Buffer FD : 5µL
+** H2O : 33µL
+** DNA : 10µL
+** XBal FD : 1µL
+** PstI FD : 1µL
+We incubated the digestion at 37°C during 15 minutes.
+===='''2 - Electrophoresis to check the digestion of BBa_K1155000 by SpeI/PstI, BBa_K1155007 and BBa_K1155003 by XBalI/PstI'''====
+Nadia
 {|
-| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.jpg|350px]]
+| style="width:350px;border:1px solid black;" |[[File:Psgel11208.jpg]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
-'''Picture: lysed cells comparison.'''
+* Well 1 : 6µL DNA Ladder
-* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
+* Well 2 : 5µL BBa_K1155000 digested by SpeI/PstI + 1µl of 6X loading dye
-* 50µl phage: the petri dish is clear, bacteria are lysed by phages.
+* Well 3 : 5µL BBa_K1155007 digested by XBaI/PstI + 1µl of 6X loading dye
-*We used a wild type strain to keep a stock and a Δ ''fnr E. coli'' strain to obtain phages that might have encapsidated a Δfnr::Km fragment.
+* Well 4 : 5µL BBa_K1155003 digested by xBaI/PstI + 1µl of 6X loading dye
+* Gel : 0.8%
 |}
-µl,50µl and 100µl petri dishes are clear so phages are multiplied.
+Expected sizes :
+* Pndh* : 111bp
-We let the antibiotic over night to select the right strain.
+* RBS_LacZ_Term : 3500 kb
+* RBS_AmilCP_Term : 824 bp
+* pSB1C3 : 2070bp
-===='''2 - Obtaining RBS_lacZ_ter. '''====
+{|
-Abdou
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We can't see any band for BBa_K1155000 digestion. The digestion failed. We will do it again. We obtain RBS-LacZ-Term and RBS-AmilCP-Term fragments. The digestion was good. We will purify it.
+|}
-Clone 10,14 and 15 plamid extraction using nucleospin kit.
+===='''3 - Digestion of BBa_K1155000 by Spe I/Pst I'''====
-Protocol : [[Team:Paris_Saclay/Protocols/hight copy plamid extraction|hight copy plamid extraction]]
+Anaïs, Nadia
+Used quantities :
-Results: concentration measured by nanodrop
+* Buffer FD : 5µL
-*clone 10: C=38ng/µl   260/280= 1.78
+* H2O : 38µL
-*clone 14: C=48.5ng/µl   260/280=1.90
+* DNA : 5µL
-*clone 15: C=52 ng/µl     260/280=1.78
+* SpeI FD : 1µL
-We still have to sequence plasmids in order to verify our results.
+* PstI FD : 1µL
-==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
+We incubate the digestion at 37°C during 15 minutes.
-===='''1 - Gibson assembly.'''====
-August 1st PCR purification to be sure about the experiment.
-BphR2 part1, BphR2 part2 and RBS_BphR2_part1, FNR part1, FNR part2 and RBS_FNR_part1.
-Protocol : [[Team:Paris_Saclay/Protocols/PCR_clean_up|PCR_clean_up]]
+==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
-Results:
+===='''Objective : obtaining FNR and BphR2 proteins'''====
+===='''1 - Electrophoresis of PCR products : RBS-BphR2 Part I, BphR2 Part I, BphR2 Part II, RBS-FNR Part I, FNR Part I and FNR Part II '''====
+Damir
 {|
-| style="width:350px;border:1px solid black;" | IMAGE
+| style="width:350px;border:1px solid black;" |[[File:Psgel21208.jpg]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
-*Well 1 : 6µL DNA Ladder
+* Well 1 : 6µL DNA Ladder
-*Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
+* Well 2 : 5µL RBS-BphR2 Part I +1µl of 6X loading dye
-*Well 3 : 5µL of BphR2 part2+1µl of 6X loading dy
+* Well 3 : 5µL BphR2 Part II +1µl of 6X loading dye
-*Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
+* Well 4 : 5µL FNR Part I +1µl of 6X loading dye
-*Well 5 : 5µL of FNR part1+1µl of 6X loading dy
+* Well 5 : 5µL FNR Part II +1µl of 6X loading dye
-*Well 6 : 5µL of FRN part2+1µl of 6X loading dy
+* Well 6 : 5µL RBS-FNR Part I +1µl of 6X loading dye
-*Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
+* Well 7 : 5µL BphR2 Part I +1µl of 6X loading dye
-*Gel : 0.8%
+* Gel : 0.8%
+|}
+Expected size
+* RBS-BphR2 Part I : 197 kb
+* BphR2 Part II : 790 kb
+* FNR Part I : 597 kb
+* FNR Part II : 200 kb
+* RBS-FNR Part I : 615 kb
+* BphR2 Part I : 178 kb
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We can't see FNR Part I, FNR Part II and BphR2 Part I fragments at the good size. We will make the PCR again. We obtain RBS-BphR2 Part I, BphR2 Part II, RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it.
+|}
+{| border="1" align="center"
+|[[Team:Paris Saclay/Notebook/August/9|<big>Previous day</big>]]
+|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
+|[[Team:Paris Saclay/Notebook/August/13|<big>Next day</big>]]
 |}
-we have no fragment so we must do again these PCRs
 {{Team:Paris_Saclay/incl_fin}}

Team:Paris Saclay/Notebook/August/12

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