Team:Paris Saclay/Notebook/August/19

From 2013.igem.org

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
-
===='''Objective : to obtain ... '''====
+
===='''Objective : obtaining BBa_K1155007'''====
-
=====1 - Gel purification of the digestion of Bba_K1155003 and Bba_K1155007 by XBaI/PstI=====
+
====1 - Sequence analysis of BBa_K1155007====
-
Nadia
+
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
N'Guyen
-
 
+
-
Nanodrop :
+
-
* RBS-LacZ-Term : 59.6ng/µL
+
-
* RBS-AmilCP-Term : 21.3ng/µL
+
{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
Concentrations are very low so we try to precipitate the DNA again.
+
Our sequence analysis was good. We obtain our biobrick BBa_K1155007.
|}
|}
-
=====2 - EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term=====
+
===='''Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K155005, BBa_K1155006 '''====
-
Nadia, XiaoJing
+
-
Protocol : [[Team:Paris_Saclay/Protocols/EtOH precipitation|EtOH precipitation]] Protocole ou à rédiger ici ?????????
+
====1 - EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term====
 +
 
 +
Nadia
 +
 
 +
Protocol : [[Team:Paris_Saclay/ethanol|EtOH precipitation]]  
We used 20µL of DNA.
We used 20µL of DNA.
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
CONCLUSION
+
The precipitation was good. We will make ligations.
|}
|}
-
=====3 - Ligation of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3=====
+
====2 - Ligation of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3====
XiaoJing  
XiaoJing  
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* NirB and RBS-LacZ-Term :
* NirB and RBS-LacZ-Term :
-
* NirB : 3µL
+
** NirB : 3µL
-
* RBS-LacZ-Term : 1µL
+
** RBS-LacZ-Term : 1µL
-
* Buffer : 2µL
+
** Buffer : 2µL
-
* H2O : 13µL
+
** H2O : 13µL
-
* Ligase : 1µL
+
** Ligase : 1µL
-
 
+
* NirB and RBS-AmilCP-Term :
* NirB and RBS-AmilCP-Term :
-
* NirB : 3µL
+
** NirB : 3µL
-
* RBS-AmilCP-Term : 1µL
+
** RBS-AmilCP-Term : 1µL
-
* Buffer : 2µL
+
** Buffer : 2µL
-
* H2O : 13µL
+
** H2O : 13µL
-
* Ligase : 1µL
+
** Ligase : 1µL
-
 
+
* NarG and RBS-LacZ-Term :
* NarG and RBS-LacZ-Term :
-
* NarG : 3µL
+
** NarG : 3µL
-
* RBS-LacZ-Term : 1µL  
+
** RBS-LacZ-Term : 1µL  
-
* Buffer : 2µL
+
** Buffer : 2µL
-
* H2O : 13µL
+
** H2O : 13µL
-
* Ligase : 1µL
+
** Ligase : 1µL
-
 
+
* NarG and RBS-AmilCP-Term :
* NarG and RBS-AmilCP-Term :
-
* NarG : 3µL
+
** NarG : 3µL
-
* RBS-AmilCP-Term : 1µL
+
** RBS-AmilCP-Term : 1µL
-
* Buffer : 2µL
+
** Buffer : 2µL
-
* H2O : 13µL
+
** H2O : 13µL
-
* Ligase : 1µL
+
** Ligase : 1µL
-
 
+
* NarK and RBS-LacZ-Term :
* NarK and RBS-LacZ-Term :
-
* Nar K : 3µL
+
** Nar K : 3µL
-
* RBS-LacZ-Term : 1µL
+
** RBS-LacZ-Term : 1µL
-
* Buffer : 2µL
+
** Buffer : 2µL
-
* H2O : 13µL
+
** H2O : 13µL
-
* Ligase : 1µL
+
** Ligase : 1µL
-
 
+
* NarK and RBS-AmilCP-Term :
* NarK and RBS-AmilCP-Term :
-
* NarK : 3µL
+
** NarK : 3µL
-
* RBS-AmilCP-Term : 1µL
+
** RBS-AmilCP-Term : 1µL
-
* Buffer : 2µL
+
** Buffer : 2µL
-
* H2O : 13µL
+
** H2O : 13µL
-
* Ligase : 1µL
+
** Ligase : 1µL
 +
 
 +
====3 - Transformation of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α====
 +
 
 +
XiaoJing
 +
 
 +
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
 +
 
 +
====4- Make competent cells of strain MG1655Z1 Δfnr::Km====
 +
 
 +
XiaoJing
 +
 
 +
Protocol : [[Team:Paris_Saclay/preparation|Preparation of super competent cells]]
 +
 
 +
====5- Transformation of MG1655Z1 Δfnr::Km====
 +
 
 +
XiaoJing
 +
 
 +
We transformed pcp20 plasmid into strain MG1655Z1 Δfnr::Km. Then we spread in LB and ampicillin antibiotic plate. We need to incubate it at 30°C for two days.
 +
 
 +
Protocol : [[Team:Paris_Saclay/Protocols/Transformation|Bacterial transformation]]
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
-
===='''Objective : obtaining FNR and BphR2 proteins (Gibson assembly) '''====
+
===='''Objective : obtaining FNR and BphR2 proteins'''====
-
=====1 - Electrophoresis gel of PCR products : BphR2 Part I=====
+
====1 - Electrophoresis gel of PCR products : BphR2 Part I====
Damir
Damir
{|
{|
-
| style="width:250px;border:1px solid black;" | [[]]
+
| style="width:250px;border:1px solid black;" | [[File:PSgel11908.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 : 5µL DNA + 1µl of 6X loading dye
+
*Well 2 : 40µL BphR2 part I + 8µl of 6X loading dye
*Gel : 1%
*Gel : 1%
|}
|}
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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We obtain a frangment at the right size. We can purify it.
+
We obtained a frangment at the right size. We can purify it.
-
|}  
+
|}
-
=====2 - Gel purification of PCR products : BphR2 Part I =====
+
====2 - Gel purification of PCR products : BphR2 Part I ====
Damir
Damir
-
Protocol : [[Team:Paris_Saclay/Protocols/Gel purification|Gel purification]]
+
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :
Nanodrop :
-
* BphR2 : 57.2ng/µL
+
* BphR2 Part I: 57.2ng/µL
  {|
  {|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
CONCLUSION
+
We lost our fragment BphR2 Part I. We will make the PCR again.
|}
|}
 +
 +
=='''Lab work, Modeling, Human practices'''==
=='''Meeting with Evry'''==
=='''Meeting with Evry'''==
[[File:PSevry2.jpg]]
[[File:PSevry2.jpg]]
 +
 +
We spent our afternoon with Evry team. Each team presented its lab, modeling and human practices project. We learned informations to improve our human practices project. We discussed about safety and gave them some advices about the pills they would make.
 +
 +
{| border="1" align="center"
 +
|[[Team:Paris Saclay/Notebook/August/14|<big>Previous day</big>]]
 +
 +
|[[Team:Paris_Saclay/Notebook|<big>Back to calendar</big>]]
 +
 +
|[[Team:Paris Saclay/Notebook/August/20|<big>Next day</big>]]
 +
|}
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 01:29, 5 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 19

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining BBa_K1155007

1 - Sequence analysis of BBa_K1155007

N'Guyen

Our sequence analysis was good. We obtain our biobrick BBa_K1155007.

Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K155005, BBa_K1155006

1 - EtOH precipitation of RBS-LacZ-Term and RBS-AmilCP-Term

Nadia

Protocol : EtOH precipitation

We used 20µL of DNA.

Nanodrop :

  • RBS-LacZ-Term : 38.4ng/µL
  • RBS-AmilCP-Term : 28.7ng/µL

The precipitation was good. We will make ligations.

2 - Ligation of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3

XiaoJing

Used quantities :

  • NirB and RBS-LacZ-Term :
    • NirB : 3µL
    • RBS-LacZ-Term : 1µL
    • Buffer : 2µL
    • H2O : 13µL
    • Ligase : 1µL
  • NirB and RBS-AmilCP-Term :
    • NirB : 3µL
    • RBS-AmilCP-Term : 1µL
    • Buffer : 2µL
    • H2O : 13µL
    • Ligase : 1µL
  • NarG and RBS-LacZ-Term :
    • NarG : 3µL
    • RBS-LacZ-Term : 1µL
    • Buffer : 2µL
    • H2O : 13µL
    • Ligase : 1µL
  • NarG and RBS-AmilCP-Term :
    • NarG : 3µL
    • RBS-AmilCP-Term : 1µL
    • Buffer : 2µL
    • H2O : 13µL
    • Ligase : 1µL
  • NarK and RBS-LacZ-Term :
    • Nar K : 3µL
    • RBS-LacZ-Term : 1µL
    • Buffer : 2µL
    • H2O : 13µL
    • Ligase : 1µL
  • NarK and RBS-AmilCP-Term :
    • NarK : 3µL
    • RBS-AmilCP-Term : 1µL
    • Buffer : 2µL
    • H2O : 13µL
    • Ligase : 1µL

3 - Transformation of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in pSB1C3 in DH5α

XiaoJing

Protocol : Bacterial transformation

4- Make competent cells of strain MG1655Z1 Δfnr::Km

XiaoJing

Protocol : Preparation of super competent cells

5- Transformation of MG1655Z1 Δfnr::Km

XiaoJing

We transformed pcp20 plasmid into strain MG1655Z1 Δfnr::Km. Then we spread in LB and ampicillin antibiotic plate. We need to incubate it at 30°C for two days.

Protocol : Bacterial transformation


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

Objective : obtaining FNR and BphR2 proteins

1 - Electrophoresis gel of PCR products : BphR2 Part I

Damir

PSgel11908.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 40µL BphR2 part I + 8µl of 6X loading dye
  • Gel : 1%

Expected size :

  • BphR2 Part I : 178 kb

We obtained a frangment at the right size. We can purify it.

2 - Gel purification of PCR products : BphR2 Part I

Damir

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

Nanodrop :

  • BphR2 Part I: 57.2ng/µL

We lost our fragment BphR2 Part I. We will make the PCR again.

Lab work, Modeling, Human practices

Meeting with Evry

PSevry2.jpg

We spent our afternoon with Evry team. Each team presented its lab, modeling and human practices project. We learned informations to improve our human practices project. We discussed about safety and gave them some advices about the pills they would make.

Previous day Back to calendar Next day

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