Paris/October 8

From 2007.igem.org

< Paris(Difference between revisions)
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(→Transformation)
 
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[[Paris/October 7|yesterday]] -- [[Paris/October 9|tomorrow]] <br>
== Digestion products ==
== Digestion products ==
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D79
| style="background: #ccffcc;" |D79
-
|
+
|pJ23100>>lox71-B0015(T)
|L44.1
|L44.1
|Xba1
|Xba1
|Pst1
|Pst1
|
|
-
|
+
|BI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D80
| style="background: #ccffcc;" |D80
-
|
+
|pJ23100>>lox71-B0015(T)
|L44.2
|L44.2
|Xba1
|Xba1
|Pst1
|Pst1
|
|
-
|
+
|BI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D81
| style="background: #ccffcc;" |D81
-
|
+
|pJ23107>>lox71-B0015(T)
|L45.1
|L45.1
|Xba1
|Xba1
|Pst1
|Pst1
|
|
-
|
+
|BI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D82
| style="background: #ccffcc;" |D82
-
|
+
|pJ23107>>lox71-B0015(T)
|L45.2
|L45.2
|Xba1
|Xba1
|Pst1
|Pst1
|
|
-
|
+
|BI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D83
| style="background: #ccffcc;" |D83
-
|
+
|pTet>>lox71-B0015(T)
|L46.1
|L46.1
|Xba1
|Xba1
|Pst1
|Pst1
|
|
-
|
+
|BI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D84
| style="background: #ccffcc;" |D84
-
|
+
|araC/pBad>>lox71-B0015(T)
|L47.1
|L47.1
|Xba1
|Xba1
|Pst1
|Pst1
|
|
-
|
+
|BI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D85
| style="background: #ccffcc;" |D85
-
|
+
|B0015(T)-lox66-RBSDapAcoli
|L53.7
|L53.7
|EcoRI
|EcoRI
|SpeI
|SpeI
|
|
-
|
+
|FI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D86
| style="background: #ccffcc;" |D86
-
|
+
|attB-lox66-RBSDapAcoli
|L54.3
|L54.3
|EcoRI
|EcoRI
|SpeI
|SpeI
|
|
-
|
+
|FI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D87
| style="background: #ccffcc;" |D87
-
|
+
|attB-lox66-RBSDapAcoli
|L54.4
|L54.4
|EcoRI
|EcoRI
|SpeI
|SpeI
|
|
-
|
+
|FI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D88
| style="background: #ccffcc;" |D88
-
|
+
|B0015(T)-lox66-RBSdapAsubtilis
|L56.3
|L56.3
|EcoRI
|EcoRI
|SpeI
|SpeI
|
|
-
|
+
|FI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D89
| style="background: #ccffcc;" |D89
-
|
+
|attB-lox66-RBSdapAsubtilis
|L57.1
|L57.1
|EcoRI
|EcoRI
|SpeI
|SpeI
|
|
-
|
+
|FI
|
|
|
|
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|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D90
| style="background: #ccffcc;" |D90
-
|
+
|attB-lox66-RBSdapAsubtilis
|L57.3
|L57.3
|EcoRI
|EcoRI
|SpeI
|SpeI
|
|
-
|
+
|FI
-
|
+
-
|
+
-
|
+
-
|- style="background: #cccccc;" 
+
-
| style="background: #ccffcc;" |D91
+
-
|
+
-
|MP14.1
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-
|EcoRI
+
-
|XbaI
+
-
|
+
-
|
+
-
|
+
-
|
+
-
|
+
-
|- style="background: #cccccc;" 
+
-
| style="background: #ccffcc;" |D92
+
-
|
+
-
|MP14.2
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-
|EcoRI
+
-
|XbaI
+
-
|
+
-
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+
-
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+
-
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-
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+
-
|- style="background: #cccccc;" 
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-
| style="background: #ccffcc;" |D93
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-
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-
|MP14.1
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-
|Spe1
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-
|Pst1
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-
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+
-
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-
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-
|
+
-
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-
|- style="background: #cccccc;" 
+
-
| style="background: #ccffcc;" |D94
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-
|
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-
|MP14.2
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-
|Spe1
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-
|Pst1
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-
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== Ligation ==
== Ligation ==
 +
 +
I should have perform ligations between those digestions upthere with the digered part [Frt-CmR-Frt] so as to be able to do Wanner later, but taking into consideration that this part had never beared any Cm resistance gene, ligations have to be posponed till this same part be ready to use.
== Transformation ==
== Transformation ==
 +
 +
i transformed into DH5α the ligation products from last Friday. Ligations had stayed in the fridged at 4°C over the weekend.
== Cultures of FR781 for pDapA caracterisation by FACS ==
== Cultures of FR781 for pDapA caracterisation by FACS ==
 +
 +
On tomorrow will be a new session for FACS mesurement fo the pDapA activity in FR781 bacteria.<br>
 +
i launched several cultures with different DAP concentrations in it : <br>
 +
<br>
 +
Mesurements will be done on both cultures at stationary phase and logarithmic phase : <br>
 +
Bacteria are pushed to enter into log phase by 1/100 dilution from stat phase LB culture to minimal medium M9.
 +
<br>
 +
Stat phase :<br>
 +
- 1mM DAP<br>
 +
- 300mM DAP<br>
 +
- 100mM DAP<br>
 +
<br>
 +
Log phase : <br>
 +
- 1mM DAP<br>
 +
- 300mM DAP<br>
 +
- 100mM DAP<br>
 +
The following have been lauched from a 300mM DAP culture at stat phase<br>
 +
- 50mM DAP<br>
 +
- 20mM DAP<br>
 +
- 0mM DAP<br>
== Culture of FX85 for future competent transformation and then recombination rate caracterisation ==
== Culture of FX85 for future competent transformation and then recombination rate caracterisation ==
 +
 +
5mL of culture with thymine and Kanamycine

Latest revision as of 14:26, 11 October 2007

yesterday -- tomorrow

Contents

Digestion products

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
D79 pJ23100>>lox71-B0015(T) L44.1 Xba1 Pst1 BI
D80 pJ23100>>lox71-B0015(T) L44.2 Xba1 Pst1 BI
D81 pJ23107>>lox71-B0015(T) L45.1 Xba1 Pst1 BI
D82 pJ23107>>lox71-B0015(T) L45.2 Xba1 Pst1 BI
D83 pTet>>lox71-B0015(T) L46.1 Xba1 Pst1 BI
D84 araC/pBad>>lox71-B0015(T) L47.1 Xba1 Pst1 BI
D85 B0015(T)-lox66-RBSDapAcoli L53.7 EcoRI SpeI FI
D86 attB-lox66-RBSDapAcoli L54.3 EcoRI SpeI FI
D87 attB-lox66-RBSDapAcoli L54.4 EcoRI SpeI FI
D88 B0015(T)-lox66-RBSdapAsubtilis L56.3 EcoRI SpeI FI
D89 attB-lox66-RBSdapAsubtilis L57.1 EcoRI SpeI FI
D90 attB-lox66-RBSdapAsubtilis L57.3 EcoRI SpeI FI

Ligation

I should have perform ligations between those digestions upthere with the digered part [Frt-CmR-Frt] so as to be able to do Wanner later, but taking into consideration that this part had never beared any Cm resistance gene, ligations have to be posponed till this same part be ready to use.

Transformation

i transformed into DH5α the ligation products from last Friday. Ligations had stayed in the fridged at 4°C over the weekend.

Cultures of FR781 for pDapA caracterisation by FACS

On tomorrow will be a new session for FACS mesurement fo the pDapA activity in FR781 bacteria.
i launched several cultures with different DAP concentrations in it :

Mesurements will be done on both cultures at stationary phase and logarithmic phase :
Bacteria are pushed to enter into log phase by 1/100 dilution from stat phase LB culture to minimal medium M9.
Stat phase :
- 1mM DAP
- 300mM DAP
- 100mM DAP

Log phase :
- 1mM DAP
- 300mM DAP
- 100mM DAP
The following have been lauched from a 300mM DAP culture at stat phase
- 50mM DAP
- 20mM DAP
- 0mM DAP

Culture of FX85 for future competent transformation and then recombination rate caracterisation

5mL of culture with thymine and Kanamycine

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