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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'''===
-===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
+===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006'''====
 ===='''1 - Tranduction of Km in MG1655Z1 ====
-Abdou, Anaïs, Damir, Nadia, XioaJing
+Abdou, Anaïs, Damir, Nadia, XiaoJing
 {|
 | style="border:1px solid black;padding:5px;background-color:#DE;" |
-We observed lysis areas. We will continue the transduction protocol.
+We observed lysis areas in the strain MG1655Z1 Δfnr::Km after transduction. We will continue the transduction protocol.
 |}
@@ Line 26: / Line 26: @@
 |}
-Protocol : [[Team:Paris_Saclay/Protocols/transduction|Transduction]]
+===='''Objective : obtaining BBa_K1155007'''====
-Our Mutant bacteria was called BW : Δfnr::Km.
+====1 - Extraction of BBa_K115007 from DH5αstrain====
-Our wild type bacteria was called MG1655Z1.
-We used kanamycine antibiotic.
-===='''Objective : obtaining Bba_K1155007'''====
-====1 - Extraction of Bba_K115007 from DH5α====
 Abdou
-Protocol : [[Team:Paris_Saclay/extraction|Hight copy plamid extraction]]
+Protocol : [[Team:Paris_Saclay/extraction|High-copy plamid extraction]]
-We used colonies number 10, 14 and 15.
+We extracted plamid from colonies number 10, 14 and 15.
 Nanodrop
-* Bba_K1155007 in clone 10 : 38ng/µl
+* BBa_K1155007 in clone 10 : 38ng/µl
-* Bba_K1155007 in clone 14 : 48.5ng/µl
+* BBa_K1155007 in clone 14 : 48.5ng/µl
-* Bba_K1155007 in clone 15 : 52 ng/µl
+* BBa_K1155007 in clone 15 : 52 ng/µl
 {|
 | style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-The extraction was good. We will sequence our plasmid.
+The extraction was good. We will sequence our plasmids.
 |}
@@ Line 59: / Line 53: @@
 {|
-| 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 Part I+1µl of 6X loading dye
+*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 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 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 5 : 5µL of FNR Part I + 1µl of 6X loading dye
-*Well 6 : 5µL of FRN Part II+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
+*Well 7: 5µL of RBS-FNR Part I + 1µl of 6X loading dye
 *Gel : 0.8%
 |}
 Expected size :
-* BphR2 Part I :
+* BphR2 Part I : 178 bp
-* BphR2 Part II :
+* BphR2 Part II : 790bp
-* RBS-BphR2 Part I :
+* RBS-BphR2 Part I : 197bp
-* FNR Part I :
+* FNR Part I : 597 bp
-* FNR Part II :
+* FNR Part II : 200bp
-* RBS-FNR PartI :
+* RBS-FNR PartI : 615bp
 {|

Team:Paris Saclay/Notebook/August/9