Team:Paris Saclay/Notebook/August/9

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''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.
 +
|}
{|
{|
| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.jpg|350px]]
| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.jpg|350px]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
'''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 : [[Team:Paris_Saclay/extraction|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 
 +
 +
{|
 +
| 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'''===
 +
 +
====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'''====
 +
 +
{|
 +
| style="width:350px;border:1px solid black;" |[[File:Psgel10908.jpg]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
*Well 1 : 6µL DNA Ladder
*Well 1 : 6µL DNA Ladder
-
*Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
+
*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%
*Gel : 0.8%
|}
|}
-
Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.
+
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
-
''2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel''
+
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We lost all our PCR fragments. We will do the PCR again.
 +
|}
-
Nadia
+
===='''2 - PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I'''====
-
Protocol : [[Team:Paris_Saclay/Protocols/Electroelution|Electroelution]]
+
Anaïs, Damir, Nadia, XiaoJing
-
We let the plasmid precipitate during the night.
+
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
-
===='''Obtaining RBS_LacZ+Term_PSB1C3'''====
+
* Bphr2 Part II :
 +
** Oligo 56F : 1µL
 +
** Oligo 57R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Pseudomonas pseudoalcaligenes'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
-
=====1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies=====
+
* RBS-Bphr2 Part I :
-
Anaïs
+
** Oligo 58F : 1µL
 +
** Oligo 57R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Pseudomonas pseudoalcaligenes'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
-
*Colony counting :
+
* FNR Part I :  
-
**Low concentration petri dish : 47 colonies
+
** Oligo 59F : 1µL
-
**High concentration petri dish : 145 colonies
+
** Oligo 60R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Escherichia coli'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
-
*Picking of 25 colonies
+
* FNR Part II :
 +
** Oligo 61F : 1µL
 +
** Oligo 62R : 1µL
 +
** Buffer Phusion : 10µL
 +
** DNA ''Escherichia coli'' : 1µL
 +
** dNTP : 1µL
 +
** Phusion : 0.5µL
 +
** H2O : 35.5µL
-
*Preparation of 700µL of Master mix
+
* RBS-FNR Part I :
-
**H<sub>2</sub>O : 590µL
+
** Oligo 63F : 1µL
-
**dNTP : 28µL
+
** Oligo 62R : 1µL
-
**VF2 primer : 3.5µL
+
** Buffer Phusion : 10µL
-
**VR primer : 3.5µL
+
** DNA ''Escherichia coli'' : 1µL
-
**DreamTaq buffer 10x : 70µL
+
** dNTP : 1µL
-
**DreamTaq enzyme : 5µL
+
** Phusion : 0.5µL
 +
** H2O : 35.5µL
-
Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
+
PCR Program :  
-
PCR Program :
+
* BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :  
-
  [IMAGE]
+
-
=====2 - Gel electrophoresis of the colony PCR products=====
+
[[File:PsPCRBphR23007.jpg|400px]]
-
Anaïs, Damir
+
-
{|
+
* FNR Part I, FNR Part II, RBS-FNR Part I :  
-
| style="width:350px;border:1px solid black;" | [[File:PsNBa8_colonies.jpg|350px]]
+
-
| style="width:350px;border:1px solid black;vertical-align:top;" |
+
-
*6µL DNA Ladder
+
-
*10µL sample per well
+
-
*Gel : 0.8%
+
-
|}
+
-
Expected size : 3583bp
+
[[File:PsPCRFNR3007.jpg|400px]]
-
Colonies 10, 14, 15 exhibit plasmids with the right length.
+
 
 +
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 +
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 +
 
 +
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 +
 
 +
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 +
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Latest revision as of 15:28, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

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.

Ps908transduction.jpg

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

Psgel10908.jpg
  • 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 :

PsPCRBphR23007.jpg

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

PsPCRFNR3007.jpg


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