Team:Paris/July 31

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{{Paris/Calendar_Links|July 30|August 01}}
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{{Paris/Calendar_Links|July 30|August 1}}
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==Oligonucleotides design==
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[[Image:RBS+.jpg|thumb|RBS +]]
 
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[[Image:RBS-.jpg|thumb|RBS -]]
 
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Creation of 3 oligonucleotides ([[team:Paris/Notebook/Oligo|O140, O141, O142]]) in order to create two particular plasmids.
 
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Those plasmids are very useful vectors to amplify promoters and to measure their forces using Standard Promoter Units.
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== Results of the transformation : Fluorescence ==
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The principle is easy to understand.
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Instead of using a big biobrick containing Promoter, RBS, GFP and Terminators, the Biobrick used for the test is only the Promoter. The standard GFP tripart (E0240) is between the biobrick site, it is just between two PstI restriction sites.
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This plasmid can be used to amplify a promoter because only the promoter is in the biobrick.
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<br><br><br><br><br><br><br><br><br><br>
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==Transformation results==
 
{| border="1"  
{| border="1"  
|align="center"|'''Ligation name'''
|align="center"|'''Ligation name'''
-
|align="center"|'''Nb of colonies'''
 
|align="center"|'''Description'''
|align="center"|'''Description'''
-
|align="center"|'''Volume gel extraction'''
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|align="center"|'''Fluorescence'''
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|-
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|align="center"|'''Comments'''
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|align="center"|L100
+
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|align="center"|0
+
-
|align="center"|rbs-TetR <-> ECFP (RBS+LVA+Term)
+
-
|align="center"|3+6
+
-
|-
+
-
|align="center"|L101
+
-
|align="center"|(1)
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|align="center"|rbs-TetR <-> GFP tripart+strong rbs
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-
|align="center"|3+6
+
|-
|-
|align="center"|L102
|align="center"|L102
-
|align="center"|+/- 100
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|align="center"|Strong rbs - YFP<br>D129 (BV) -  D118 (BI) 
-
|align="center"|strong RBS (0,6)<-> YFP
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|align="center"|No
-
|align="center"|5+10
+
|align="center"|OK
|-
|-
|align="center"|L103
|align="center"|L103
-
|align="center"|a lot ++
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|align="center"|Strong rbs - mRFP<br>D129 (BV) -  D122 (BI) 
-
|align="center"|strong RBS (0,6)<-> GFP
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|align="center"|No
-
|align="center"|5+5
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|align="center"|OK
|-
|-
|align="center"|L104
|align="center"|L104
-
|align="center"|a lot ++
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|align="center"|Strong rbs - lasR activator<br>D129 (BV) -  D114 (BI) 
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|align="center"|strong RBS (0,6)<-> LasR activator (+LVA)
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|align="center"|No
-
|align="center"|5+4
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|align="center"|OK
|-
|-
|align="center"|L105
|align="center"|L105
-
|align="center"|a lot ++
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|align="center"|Strong promoter - ECFP<br>D123 (BV) - D130 (BI)
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|align="center"|strong constitutive promoter <-> ECFP
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|align="center"|Yes (some others red because of the promoter)
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|align="center"|5+8
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|align="center"|OK
|-
|-
|align="center"|L106
|align="center"|L106
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|align="center"|a lot ++
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|align="center"|Strong promoter - gfp Tripart<br>D123 (BV) - D131 (BI)
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|align="center"|strong constitutive promoter <-> GFP tripart
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|align="center"|Yes (some others red <br> because of the promoter)
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|align="center"|5+8
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|align="center"|OK
|-
|-
|align="center"|L107
|align="center"|L107
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|align="center"|a lot ++
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|align="center"|Strongest promoter - ECFP<br>D103 (BV) - D130 (BI)
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|align="center"|strongest constitutive promoter <-> ECFP
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|align="center"|Yes (some others red <br>because of the promoter)
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|align="center"|5+8
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|align="center"|OK
|-
|-
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|align="center"|L108
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|align="center"|L108 n°2 (the right one)
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|align="center"|+/- 100 (2 dishes!?)
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|align="center"|Strong promoter -  gfp Tripart<br>D103 (BV) - D131 (BI)
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|align="center"|strongest constitutive promoter <-> GFP tripart
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|align="center"|Yes (some others red because of the promoter)
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|align="center"|5+8
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|align="center"|OK
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|-
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|align="center"|L109
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|align="center"|+/- 30 or a lot ++ (2 dishes !?)  
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|align="center"|medium constitutive promoter <-> ECFP
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|align="center"|4+7
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|-
|-
|align="center"|L110
|align="center"|L110
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|align="center"|a lot ++
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|align="center"|Medium promoter - gfp Tripart<br>D124 (BV) - D131 (BI)
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|align="center"|medium constitutive promoter <-> gfp tripart
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|align="center"|Yes (some others red because of the promoter)
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|align="center"|4+7
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|align="center"|OK
|-
|-
|align="center"|L111
|align="center"|L111
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|align="center"|a lot ++
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|align="center"|Weak promoter - ECFP<br>D104 (BV) - D130 (BI)
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|align="center"|weak constitutive promoter <-> ECFP
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|align="center"|No
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|align="center"|3+5
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|align="center"|To do again
|-
|-
|align="center"|L112
|align="center"|L112
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|align="center"|a lot ++
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|align="center"|Weak promoter - gfp tripart<br>D104 (BV) - D131 (BI)
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|align="center"|weak constitutive promoter <-> gfp tripart
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|align="center"|No
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|align="center"|3+5
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|align="center"|To do again
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|-
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|align="center"|L113
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|align="center"|1
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|align="center"|Arac pBAD <-> ECFP
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|align="center"|5+2.5
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|-
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|align="center"|L114
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|align="center"|2
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|align="center"|Arac pBAD <-> gfp tripart
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|align="center"|5+2.5
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|-
|-
|align="center"|L115
|align="center"|L115
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|align="center"|a lot ++
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|align="center"|pLas - ECFP<br> D105 (BV)- D130 (BI)
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|align="center"|pLas <-> ECFP
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|align="center"|No
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|align="center"|7+11
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|align="center"|OK
|-
|-
|align="center"|L116
|align="center"|L116
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|align="center"|a lot ++
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|align="center"|pLas - gfp Tripart<br>D105 (BV) - D131 (BI)
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|align="center"|pLas <-> gfp tripart
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|align="center"|1 green
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|align="center"|7+11
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|align="center"|To do again
|-
|-
|align="center"|L117
|align="center"|L117
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|align="center"|+/- 30
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|align="center"|yfp - Double Terminator<br>D117 (FI) - D125 (FV)
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|align="center"|Double terminator <-> YFP
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|align="center"|No
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|align="center"|4+3
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|align="center"|OK
|-
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|align="center"|L118
|align="center"|L118
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|align="center"|+/- 30
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|align="center"|rfp - Double Terminator<br>D121 (FI) - D125 (FV)
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|align="center"|Double terminator <-> mRFP
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|align="center"|No
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|align="center"|4+2
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|align="center"|OK
|-
|-
|align="center"|L119
|align="center"|L119
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|align="center"|+/- 30
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|align="center"|lasR activator + LVA - Double Terminator<br>D113 (FI) - D125 (FV)
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|align="center"|Double terminator <-> LasR activator (+LVA)
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|align="center"|No
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|align="center"|4+2
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|align="center"|OK
|-
|-
|align="center"|L120
|align="center"|L120
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|align="center"|a lot ++
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|align="center"|tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI)
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|align="center"|tetR repressible promoter <-> ECFP
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|align="center"|No
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|align="center"|6+7.5
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|align="center"|To do again
|-
|-
|align="center"|L121
|align="center"|L121
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|align="center"|a lot ++
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|align="center"|tetR repressible promoter - gfp tripart<br>D106 (BV) - D131 (BI)
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|align="center"|tetR repressible promoter <-> gfp tripart
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|align="center"|Yes (some others red because of the promoter)
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|align="center"|6+7.5
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|align="center"|OK
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|-
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|align="center"|L122
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|align="center"|0
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|align="center"|B0034rbs-LasI <-> ECFP
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|align="center"|7+3.5
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|-
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|align="center"|L123
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|align="center"|1
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|align="center"|B0034rbs-LasI <-> gfp tripart
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|align="center"|7+3.5
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|-
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|align="center"|L124
|align="center"|L124
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|align="center"|a lot ++
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|align="center"|Strongest RBS - mRFP<br>D102 (BV) - D122 (BI)
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|align="center"|strongest RBS (1) <-> GFP
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|align="center"|No
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|align="center"|6+5
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|align="center"|OK
|-
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|align="center"|L125
|align="center"|L125
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|align="center"|a lot ++
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|align="center"|Strongest RBS - YFP<br>D102 (BV) - D118 (BI)
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|align="center"|strongest RBS (1) <-> YFP
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|align="center"|No
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|align="center"|6+10
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|align="center"|OK
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|-
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|}
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|align="center"|L126
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|align="center"|no dish found!!!
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== Ligations ==
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|align="center"|strongest RBS (1) <->  activator (+LVA)
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|align="center"|6+4
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''We tried again the following ligations :
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|-
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L100, L101, L109, L113, L114, L120, L122, L123, L126, L127''
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|align="center"|L127
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|align="center"|no dish found!!!
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=== Protocol ===
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|align="center"|Double terminator <-> GFP
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|align="center"|6+3
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''For each samples,''  
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|-
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|align="center"|'''Controls'''
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* 1 µl Ligase
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|align="center"|
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* X µl Vector
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|align="center"|
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* Y µl Insert
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|align="center"|
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* 2 µl Ligase Buffer 10x
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|-
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* 20 µl qsp H2O
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|align="center"|C1
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|align="center"|0
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* Incubates O/N at 16°C (in the cold room).
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|align="center"|D110
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|align="center"|3
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|-
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=== List of new Ligation transformation ===
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|align="center"|C2
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|align="center"|5
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{| border="1"  
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|align="center"|D129
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|align=center|'''Name'''
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|align="center"|5
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|align=center|'''Vector'''
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|-
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|align=center|'''Insert'''
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|align="center"|C3
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|align=center|'''Antibio'''
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|align="center"|+/- 100
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|align=center|'''Vol. vector (µl)'''
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|align="center"|D123
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|align=center|'''Vol. insert (µl)'''
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|align="center"|5
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|align=center|'''Vol. H20 (µl)'''
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|-
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|align="center"|C4
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|style="background: #ccccff;"| L100
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|align="center"|+/- 20
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|align=center| D110
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|align="center"|D103
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|align=center| D130
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|align="center"|5
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|align=center| A
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|align=center|3
 +
|align=center|6
 +
|align=center|8
|-
|-
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|align="center"|C5
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|style="background: #ccccff;"| L101
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|align="center"|+/- 100
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|align=center| D110
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|align="center"|Double terminator <-> GFP
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|align=center| D131
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|align="center"|4
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|align=center| A
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|align=center|3
 +
|align=center|6
 +
|align=center|8
|-
|-
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|align="center"|C6
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|style="background: #ccccff;"| L109
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|align="center"|+/- 100
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|align=center| D124
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|align="center"|Double terminator <-> GFP
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|align=center| D130
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|align="center"|3
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|align=center| A
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|align=center|4
 +
|align=center|7
 +
|align=center|6
|-
|-
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|align="center"|C7
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|style="background: #ccccff;"| L113
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|align="center"|0
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|align=center| D126
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|align="center"|Double terminator <-> GFP
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|align=center| D130
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|align="center"|5
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|align=center| K
 +
|align=center|5
 +
|align=center|2.5
 +
|align=center|9.5
|-
|-
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|align="center"|C8
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|style="background: #ccccff;"| L114
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|align="center"|+/- 20
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|align=center| D126
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|align="center"|Double terminator <-> GFP
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|align=center| D131
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|align="center"|7
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|align=center| K
 +
|align=center|5
 +
|align=center|2.5
 +
|align=center|9.5
|-
|-
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|align="center"|C9
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|style="background: #ccccff;"| L120
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|align="center"|0
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|align=center| D106
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|align="center"|Double terminator <-> GFP
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|align=center| D130
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|align="center"|4
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|align=center| A
 +
|align=center|6
 +
|align=center|7.5
 +
|align=center|3.5
|-
|-
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|align="center"|C10
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|style="background: #ccccff;"| L122
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|align="center"|0
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|align=center| D107
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|align="center"|Double terminator <-> GFP
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|align=center| D130
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|align="center"|6
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|align=center| A
 +
|align=center|7
 +
|align=center|3.5
 +
|align=center|6.5
|-
|-
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|align="center"|C11
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|style="background: #ccccff;"| L123
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|align="center"|0
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|align=center| D107
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|align="center"|Double terminator <-> GFP
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|align=center| D131
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|align="center"|7
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|align=center| A
 +
|align=center|7
 +
|align=center|3.5
 +
|align=center|6.5
|-
|-
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|align="center"|C12
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|style="background: #ccccff;"| L126
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|align="center"|+/- 10
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|align=center| D102
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|align="center"|Double terminator <-> GFP
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|align=center| D114
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|align="center"|6
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|align=center| A
 +
|align=center|6
 +
|align=center|4
 +
|align=center|7
|-
|-
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|align="center"|Positive control
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|style="background: #ccccff;"| L127
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|align="center"|155 (transformation efficiency:1.5*10^7/ug)
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|align=center| D125
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|align="center"|???
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|align=center| D119
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|align="center"|
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|align=center| A
 +
|align=center|6
 +
|align=center|3
 +
|align=center|8
|}
|}

Latest revision as of 17:04, 13 August 2008

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Contents

Results of the transformation : Fluorescence

Ligation name Description Fluorescence Comments
L102 Strong rbs - YFP
D129 (BV) - D118 (BI) 		No OK
L103 Strong rbs - mRFP
D129 (BV) - D122 (BI) 		No OK
L104 Strong rbs - lasR activator
D129 (BV) - D114 (BI) 		No OK
L105 Strong promoter - ECFP
D123 (BV) - D130 (BI) 		Yes (some others red because of the promoter) OK
L106 Strong promoter - gfp Tripart
D123 (BV) - D131 (BI) 		Yes (some others red
because of the promoter) 		OK
L107 Strongest promoter - ECFP
D103 (BV) - D130 (BI) 		Yes (some others red
because of the promoter) 		OK
L108 n°2 (the right one) Strong promoter - gfp Tripart
D103 (BV) - D131 (BI) 		Yes (some others red because of the promoter) OK
L110 Medium promoter - gfp Tripart
D124 (BV) - D131 (BI) 		Yes (some others red because of the promoter) OK
L111 Weak promoter - ECFP
D104 (BV) - D130 (BI) 		No To do again
L112 Weak promoter - gfp tripart
D104 (BV) - D131 (BI) 		No To do again
L115 pLas - ECFP
D105 (BV)- D130 (BI) 		No OK
L116 pLas - gfp Tripart
D105 (BV) - D131 (BI) 		1 green To do again
L117 yfp - Double Terminator
D117 (FI) - D125 (FV) 		No OK
L118 rfp - Double Terminator
D121 (FI) - D125 (FV) 		No OK
L119 lasR activator + LVA - Double Terminator
D113 (FI) - D125 (FV) 		No OK
L120 tetR repressible promoter - ECFP
D106 (BV) - D130 (BI) 		No To do again
L121 tetR repressible promoter - gfp tripart
D106 (BV) - D131 (BI) 		Yes (some others red because of the promoter) OK
L124 Strongest RBS - mRFP
D102 (BV) - D122 (BI) 		No OK
L125 Strongest RBS - YFP
D102 (BV) - D118 (BI) 		No OK

Ligations

We tried again the following ligations : L100, L101, L109, L113, L114, L120, L122, L123, L126, L127

Protocol

For each samples,

  • 1 µl Ligase
  • X µl Vector
  • Y µl Insert
  • 2 µl Ligase Buffer 10x
  • 20 µl qsp H2O
  • Incubates O/N at 16°C (in the cold room).


List of new Ligation transformation

Name Vector Insert Antibio Vol. vector (µl) Vol. insert (µl) Vol. H20 (µl)
L100 D110 D130 A 3 6 8
L101 D110 D131 A 3 6 8
L109 D124 D130 A 4 7 6
L113 D126 D130 K 5 2.5 9.5
L114 D126 D131 K 5 2.5 9.5
L120 D106 D130 A 6 7.5 3.5
L122 D107 D130 A 7 3.5 6.5
L123 D107 D131 A 7 3.5 6.5
L126 D102 D114 A 6 4 7
L127 D125 D119 A 6 3 8
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