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Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006

1 - Tranduction of Km in MG1655Z1

Abdou, Anaïs, Damir, Nadia, XiaoJing

We observed lysis areas in the strain MG1655Z1 Δfnr::Km after transduction. We will continue the transduction protocol.

Picture: lysed cells comparison.

0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
50µl phage: the petri dish is clear, bacteria are lysed by phages.

Objective : obtaining BBa_K1155007

1 - Extraction of BBa_K115007 from DH5αstrain

Abdou

Protocol : High-copy plamid extraction

We extracted plamid from colonies number 10, 14 and 15.

Nanodrop

BBa_K1155007 in clone 10 : 38ng/µl
BBa_K1155007 in clone 14 : 48.5ng/µl
BBa_K1155007 in clone 15 : 52 ng/µl

The extraction was good. We will sequence our plasmids.

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

Objective : Obtaining FNR and BphR2 proteins

1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification

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

Expected size :

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

We lost all our PCR fragments. We will do the PCR again.

2 - PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I

Anaïs, Damir, Nadia, XiaoJing

Used quantities :

Bphr2 Part I :
- Oligo 54F : 1µL
- Oligo 55R : 1µL
- Buffer Phusion : 10µL
- DNA of Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

Bphr2 Part II :
- Oligo 56F : 1µL
- Oligo 57R : 1µL
- Buffer Phusion : 10µL
- DNA Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

RBS-Bphr2 Part I :
- Oligo 58F : 1µL
- Oligo 57R : 1µL
- Buffer Phusion : 10µL
- DNA Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

FNR Part I :
- Oligo 59F : 1µL
- Oligo 60R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

FNR Part II :
- Oligo 61F : 1µL
- Oligo 62R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

RBS-FNR Part I :
- Oligo 63F : 1µL
- Oligo 62R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL

PCR Program :

BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :

FNR Part I, FNR Part II, RBS-FNR Part I :

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@@ Line 7: / Line 7: @@
 ==='''A - Aerobic/Anaerobic regulation system'''===
-===='''1 - Obtaining Δ ''fnr E. coli'' strain by transduction to test our biobricks'''====
+===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
-Abdou, Anais, Damir, Nadia, XiaoJing
+===='''1 - Tranduction of Km in MG1655Z1 ====
-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)
+Abdou, Anaïs, Damir, Nadia, XiaoJing
-Protocol : [[Team:Paris_Saclay/Protocols/transduction|transduction]]
+{|
+| style="border:1px solid black;padding:5px;background-color:#DE;" |
+We observed lysis areas in the strain MG1655Z1 Δfnr::Km after transduction. We will continue the transduction protocol.
+|}
 {|
@@ Line 21: / Line 24: @@
 * 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
 * 50µl phage: the petri dish is clear, bacteria are lysed by phages.
-*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.
 |}
-µl,50µl and 100µl petri dishes are clear so phages are multiplied.
+===='''Objective : obtaining BBa_K1155007'''====
-We let the antibiotic over night to select the right strain.
+====1 - Extraction of BBa_K115007 from DH5αstrain====
-===='''2 - Obtaining RBS_lacZ_ter. '''====
 Abdou
-Clone 10,14 and 15 plamid extraction using nucleospin kit.
+Protocol : [[Team:Paris_Saclay/extraction|High-copy plamid extraction]]
-Protocol : [[Team:Paris_Saclay/Protocols/hight copy plamid extraction|hight copy plamid extraction]]
+We extracted plamid from colonies number 10, 14 and 15.
-Results: concentration measured by nanodrop
+Nanodrop
-*clone 10: C=38ng/µl   260/280= 1.78
+* BBa_K1155007 in clone 10 : 38ng/µl
-*clone 14: C=48.5ng/µl   260/280=1.90
+* BBa_K1155007 in clone 14 : 48.5ng/µl
-*clone 15: C=52 ng/µl     260/280=1.78
+* BBa_K1155007 in clone 15 : 52 ng/µl
-We still have to sequence plasmids in order to verify our results.
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+The extraction was good. We will sequence our plasmids.
+|}
 ==='''A - Aerobic/Anaerobic regulation system / B - PCB sensing system'''===
-===='''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]]
+====Objective : Obtaining FNR and BphR2 proteins====
-Results:
+===='''1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification'''====
 {|
-| style="width:350px;border:1px solid black;" | IMAGE
+| style="width:350px;border:1px solid black;" |[[File:Psgel10908.jpg]]
 | style="width:350px;border:1px solid black;vertical-align:top;" |
 *Well 1 : 6µL DNA Ladder
-*Well 2 : 5µL of BphR2 part1+1µl of 6X loading dy
+*Well 2 : 5µL of BphR2 Part I + 1µl of 6X loading dye
-*Well 3 : 5µL of BphR2 part2+1µl of 6X loading dy
+*Well 3 : 5µL of BphR2 Part II + 1µl of 6X loading dye
-*Well 4 : 5µL of RBS_BphR2 part1+1µl of 6X loading dy
+*Well 4 : 5µL of RBS-BphR2 Part I + 1µl of 6X loading dye
-*Well 5 : 5µL of FNR part1+1µl of 6X loading dy
+*Well 5 : 5µL of FNR Part I + 1µl of 6X loading dye
-*Well 6 : 5µL of FRN part2+1µl of 6X loading dy
+*Well 6 : 5µL of FRN Part II + 1µl of 6X loading dye
-*Well 7: 5µL of RBS_FNR part1+1µl of 6X loading dy
+*Well 7: 5µL of RBS-FNR Part I + 1µl of 6X loading dye
 *Gel : 0.8%
 |}
-we have no fragment so we must do again these PCRs
+Expected size :
+* BphR2 Part I : 178 bp
+* BphR2 Part II : 790bp
+* RBS-BphR2 Part I : 197bp
+* FNR Part I : 597 bp
+* FNR Part II : 200bp
+* RBS-FNR PartI : 615bp
+{|
+| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
+We lost all our PCR fragments. We will do the PCR again.
+|}
+===='''2 - PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I'''====
+Anaïs, Damir, Nadia, XiaoJing
+Used quantities :
+* Bphr2 Part I :
+** Oligo 54F : 1µL
+** Oligo 55R : 1µL
+** Buffer Phusion : 10µL
+** DNA of ''Pseudomonas pseudoalcaligenes'' : 1µL
+** dNTP : 1µL
+** Phusion : 0.5µL
+** H2O : 35.5µL
+* Bphr2 Part II :
+** Oligo 56F : 1µL
+** Oligo 57R : 1µL
+** Buffer Phusion : 10µL
+** DNA ''Pseudomonas pseudoalcaligenes'' : 1µL
+** dNTP : 1µL
+** Phusion : 0.5µL
+** H2O : 35.5µL
+* RBS-Bphr2 Part I :
+** Oligo 58F : 1µL
+** Oligo 57R : 1µL
+** Buffer Phusion : 10µL
+** DNA ''Pseudomonas pseudoalcaligenes'' : 1µL
+** dNTP : 1µL
+** Phusion : 0.5µL
+** H2O : 35.5µL
+* FNR Part I :
+** Oligo 59F : 1µL
+** Oligo 60R : 1µL
+** Buffer Phusion : 10µL
+** DNA ''Escherichia coli'' : 1µL
+** dNTP : 1µL
+** Phusion : 0.5µL
+** H2O : 35.5µL
+* FNR Part II :
+** Oligo 61F : 1µL
+** Oligo 62R : 1µL
+** Buffer Phusion : 10µL
+** DNA ''Escherichia coli'' : 1µL
+** dNTP : 1µL
+** Phusion : 0.5µL
+** H2O : 35.5µL
+* RBS-FNR Part I :
+** Oligo 63F : 1µL
+** Oligo 62R : 1µL
+** Buffer Phusion : 10µL
+** DNA ''Escherichia coli'' : 1µL
+** dNTP : 1µL
+** Phusion : 0.5µL
+** H2O : 35.5µL
+PCR Program :
+* BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :
+[[File:PsPCRBphR23007.jpg|400px]]
+* FNR Part I, FNR Part II, RBS-FNR Part I :
+[[File:PsPCRFNR3007.jpg|400px]]
+{| border="1" align="center"
+|[[Team:Paris Saclay/Notebook/August/8|<big>Previous day</big>]]
+|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
+|[[Team:Paris Saclay/Notebook/August/12|<big>Next day</big>]]
+|}
 {{Team:Paris_Saclay/incl_fin}}

Team:Paris Saclay/Notebook/August/9