Team:Paris/July 31

From 2008.igem.org

(Difference between revisions)
(→Oligonucleotides design)
(→Results of the transformation : Fluorescence)
 
(19 intermediate revisions not shown)
Line 1: Line 1:
-
{{Paris/Calendar_Links|July 30|August 01}}
+
{{Paris/Calendar_Links|July 30|August 1}}
-
==Transformation results==
+
== Results of the transformation : Fluorescence ==
 +
 
{| 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'''
+
|align="center"|'''Fluorescence'''
-
|-
+
|align="center"|'''Comments'''
-
|align="center"|L100
+
-
|align="center"|0
+
-
|align="center"|rbs-TetR <-> ECFP (RBS+LVA+Term)
+
-
|align="center"|3+6
+
-
|-
+
-
|align="center"|L101
+
-
|align="center"|(1)
+
-
|align="center"|rbs-TetR <-> GFP tripart+strong rbs
+
-
|align="center"|3+6
+
|-
|-
|align="center"|L102
|align="center"|L102
-
|align="center"|+/- 100
+
|align="center"|Strong rbs - YFP<br>D129 (BV) -  D118 (BI) 
-
|align="center"|strong RBS (0,6)<-> YFP
+
|align="center"|No
-
|align="center"|5+10
+
|align="center"|OK
|-
|-
|align="center"|L103
|align="center"|L103
-
|align="center"|a lot ++
+
|align="center"|Strong rbs - mRFP<br>D129 (BV) -  D122 (BI) 
-
|align="center"|strong RBS (0,6)<-> GFP
+
|align="center"|No
-
|align="center"|5+5
+
|align="center"|OK
|-
|-
|align="center"|L104
|align="center"|L104
-
|align="center"|a lot ++
+
|align="center"|Strong rbs - lasR activator<br>D129 (BV) -  D114 (BI) 
-
|align="center"|strong RBS (0,6)<-> LasR activator (+LVA)
+
|align="center"|No
-
|align="center"|5+4
+
|align="center"|OK
|-
|-
|align="center"|L105
|align="center"|L105
-
|align="center"|a lot ++
+
|align="center"|Strong promoter - ECFP<br>D123 (BV) - D130 (BI)
-
|align="center"|strong constitutive promoter <-> ECFP
+
|align="center"|Yes (some others red because of the promoter)
-
|align="center"|5+8
+
|align="center"|OK
|-
|-
|align="center"|L106
|align="center"|L106
-
|align="center"|a lot ++
+
|align="center"|Strong promoter - gfp Tripart<br>D123 (BV) - D131 (BI)
-
|align="center"|strong constitutive promoter <-> GFP tripart
+
|align="center"|Yes (some others red <br> because of the promoter)
-
|align="center"|5+8
+
|align="center"|OK
|-
|-
|align="center"|L107
|align="center"|L107
-
|align="center"|a lot ++
+
|align="center"|Strongest promoter - ECFP<br>D103 (BV) - D130 (BI)
-
|align="center"|strongest constitutive promoter <-> ECFP
+
|align="center"|Yes (some others red <br>because of the promoter)
-
|align="center"|5+8
+
|align="center"|OK
|-
|-
-
|align="center"|L108
+
|align="center"|L108 n°2 (the right one)
-
|align="center"|+/- 100 (2 dishes!?)
+
|align="center"|Strong promoter -  gfp Tripart<br>D103 (BV) - D131 (BI)
-
|align="center"|strongest constitutive promoter <-> GFP tripart
+
|align="center"|Yes (some others red because of the promoter)
-
|align="center"|5+8
+
|align="center"|OK
-
|-
+
-
|align="center"|L109
+
-
|align="center"|+/- 30 or a lot ++ (2 dishes !?)  
+
-
|align="center"|medium constitutive promoter <-> ECFP
+
-
|align="center"|4+7
+
|-
|-
|align="center"|L110
|align="center"|L110
-
|align="center"|a lot ++
+
|align="center"|Medium promoter - gfp Tripart<br>D124 (BV) - D131 (BI)
-
|align="center"|medium constitutive promoter <-> gfp tripart
+
|align="center"|Yes (some others red because of the promoter)
-
|align="center"|4+7
+
|align="center"|OK
|-
|-
|align="center"|L111
|align="center"|L111
-
|align="center"|a lot ++
+
|align="center"|Weak promoter - ECFP<br>D104 (BV) - D130 (BI)
-
|align="center"|weak constitutive promoter <-> ECFP
+
|align="center"|No
-
|align="center"|3+5
+
|align="center"|To do again
|-
|-
|align="center"|L112
|align="center"|L112
-
|align="center"|a lot ++
+
|align="center"|Weak promoter - gfp tripart<br>D104 (BV) - D131 (BI)
-
|align="center"|weak constitutive promoter <-> gfp tripart
+
|align="center"|No
-
|align="center"|3+5
+
|align="center"|To do again
-
|-
+
-
|align="center"|L113
+
-
|align="center"|1
+
-
|align="center"|Arac pBAD <-> ECFP
+
-
|align="center"|5+2.5
+
-
|-
+
-
|align="center"|L114
+
-
|align="center"|2
+
-
|align="center"|Arac pBAD <-> gfp tripart
+
-
|align="center"|5+2.5
+
|-
|-
|align="center"|L115
|align="center"|L115
-
|align="center"|a lot ++
+
|align="center"|pLas - ECFP<br> D105 (BV)- D130 (BI)
-
|align="center"|pLas <-> ECFP
+
|align="center"|No
-
|align="center"|7+11
+
|align="center"|OK
|-
|-
|align="center"|L116
|align="center"|L116
-
|align="center"|a lot ++
+
|align="center"|pLas - gfp Tripart<br>D105 (BV) - D131 (BI)
-
|align="center"|pLas <-> gfp tripart
+
|align="center"|1 green
-
|align="center"|7+11
+
|align="center"|To do again
|-
|-
|align="center"|L117
|align="center"|L117
-
|align="center"|+/- 30
+
|align="center"|yfp - Double Terminator<br>D117 (FI) - D125 (FV)
-
|align="center"|Double terminator <-> YFP
+
|align="center"|No
-
|align="center"|4+3
+
|align="center"|OK
|-
|-
|align="center"|L118
|align="center"|L118
-
|align="center"|+/- 30
+
|align="center"|rfp - Double Terminator<br>D121 (FI) - D125 (FV)
-
|align="center"|Double terminator <-> mRFP
+
|align="center"|No
-
|align="center"|4+2
+
|align="center"|OK
|-
|-
|align="center"|L119
|align="center"|L119
-
|align="center"|+/- 30
+
|align="center"|lasR activator + LVA - Double Terminator<br>D113 (FI) - D125 (FV)
-
|align="center"|Double terminator <-> LasR activator (+LVA)
+
|align="center"|No
-
|align="center"|4+2
+
|align="center"|OK
|-
|-
|align="center"|L120
|align="center"|L120
-
|align="center"|a lot ++
+
|align="center"|tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI)
-
|align="center"|tetR repressible promoter <-> ECFP
+
|align="center"|No
-
|align="center"|6+7.5
+
|align="center"|To do again
|-
|-
|align="center"|L121
|align="center"|L121
-
|align="center"|a lot ++
+
|align="center"|tetR repressible promoter - gfp tripart<br>D106 (BV) - D131 (BI)
-
|align="center"|tetR repressible promoter <-> gfp tripart
+
|align="center"|Yes (some others red because of the promoter)
-
|align="center"|6+7.5
+
|align="center"|OK
-
|-
+
-
|align="center"|L122
+
-
|align="center"|0
+
-
|align="center"|B0034rbs-LasI <-> ECFP
+
-
|align="center"|7+3.5
+
-
|-
+
-
|align="center"|L123
+
-
|align="center"|1
+
-
|align="center"|B0034rbs-LasI <-> gfp tripart
+
-
|align="center"|7+3.5
+
|-
|-
|align="center"|L124
|align="center"|L124
-
|align="center"|a lot ++
+
|align="center"|Strongest RBS - mRFP<br>D102 (BV) - D122 (BI)
-
|align="center"|strongest RBS (1) <-> GFP
+
|align="center"|No
-
|align="center"|6+5
+
|align="center"|OK
|-
|-
|align="center"|L125
|align="center"|L125
-
|align="center"|a lot ++
+
|align="center"|Strongest RBS - YFP<br>D102 (BV) - D118 (BI)
-
|align="center"|strongest RBS (1) <-> YFP
+
|align="center"|No
-
|align="center"|6+10
+
|align="center"|OK
-
|-
+
|}
-
|align="center"|L126
+
 
-
|align="center"|no dish found!!!
+
== Ligations ==
-
|align="center"|strongest RBS (1) <->  activator (+LVA)
+
 
-
|align="center"|6+4
+
''We tried again the following ligations :
-
|-
+
L100, L101, L109, L113, L114, L120, L122, L123, L126, L127''
-
|align="center"|L127
+
 
-
|align="center"|no dish found!!!
+
=== Protocol ===
-
|align="center"|Double terminator <-> GFP
+
 
-
|align="center"|6+3
+
''For each samples,''  
-
|-
+
 
-
|align="center"|'''Controls'''
+
* 1 µl Ligase
-
|align="center"|
+
* X µl Vector
-
|align="center"|
+
* Y µl Insert
-
|align="center"|
+
* 2 µl Ligase Buffer 10x
-
|-
+
* 20 µl qsp H2O
-
|align="center"|C1
+
 
-
|align="center"|0
+
* Incubates O/N at 16°C (in the cold room).
-
|align="center"|D110
+
 
-
|align="center"|3
+
 
-
|-
+
=== List of new Ligation transformation ===
-
|align="center"|C2
+
 
-
|align="center"|5
+
{| border="1"  
-
|align="center"|D129
+
|align=center|'''Name'''
-
|align="center"|5
+
|align=center|'''Vector'''
-
|-
+
|align=center|'''Insert'''
-
|align="center"|C3
+
|align=center|'''Antibio'''
-
|align="center"|+/- 100
+
|align=center|'''Vol. vector (µl)'''
-
|align="center"|D123
+
|align=center|'''Vol. insert (µl)'''
-
|align="center"|5
+
|align=center|'''Vol. H20 (µl)'''
|-
|-
-
|align="center"|C4
+
|style="background: #ccccff;"| L100
-
|align="center"|+/- 20
+
|align=center| D110
-
|align="center"|D103
+
|align=center| D130
-
|align="center"|5
+
|align=center| A
 +
|align=center|3
 +
|align=center|6
 +
|align=center|8
|-
|-
-
|align="center"|C5
+
|style="background: #ccccff;"| L101
-
|align="center"|+/- 100
+
|align=center| D110
-
|align="center"|Double terminator <-> GFP
+
|align=center| D131
-
|align="center"|4
+
|align=center| A
 +
|align=center|3
 +
|align=center|6
 +
|align=center|8
|-
|-
-
|align="center"|C6
+
|style="background: #ccccff;"| L109
-
|align="center"|+/- 100
+
|align=center| D124
-
|align="center"|Double terminator <-> GFP
+
|align=center| D130
-
|align="center"|3
+
|align=center| A
 +
|align=center|4
 +
|align=center|7
 +
|align=center|6
|-
|-
-
|align="center"|C7
+
|style="background: #ccccff;"| L113
-
|align="center"|0
+
|align=center| D126
-
|align="center"|Double terminator <-> GFP
+
|align=center| D130
-
|align="center"|5
+
|align=center| K
 +
|align=center|5
 +
|align=center|2.5
 +
|align=center|9.5
|-
|-
-
|align="center"|C8
+
|style="background: #ccccff;"| L114
-
|align="center"|+/- 20
+
|align=center| D126
-
|align="center"|Double terminator <-> GFP
+
|align=center| D131
-
|align="center"|7
+
|align=center| K
 +
|align=center|5
 +
|align=center|2.5
 +
|align=center|9.5
|-
|-
-
|align="center"|C9
+
|style="background: #ccccff;"| L120
-
|align="center"|0
+
|align=center| D106
-
|align="center"|Double terminator <-> GFP
+
|align=center| D130
-
|align="center"|4
+
|align=center| A
 +
|align=center|6
 +
|align=center|7.5
 +
|align=center|3.5
|-
|-
-
|align="center"|C10
+
|style="background: #ccccff;"| L122
-
|align="center"|0
+
|align=center| D107
-
|align="center"|Double terminator <-> GFP
+
|align=center| D130
-
|align="center"|6
+
|align=center| A
 +
|align=center|7
 +
|align=center|3.5
 +
|align=center|6.5
|-
|-
-
|align="center"|C11
+
|style="background: #ccccff;"| L123
-
|align="center"|0
+
|align=center| D107
-
|align="center"|Double terminator <-> GFP
+
|align=center| D131
-
|align="center"|7
+
|align=center| A
 +
|align=center|7
 +
|align=center|3.5
 +
|align=center|6.5
|-
|-
-
|align="center"|C12
+
|style="background: #ccccff;"| L126
-
|align="center"|+/- 10
+
|align=center| D102
-
|align="center"|Double terminator <-> GFP
+
|align=center| D114
-
|align="center"|6
+
|align=center| A
 +
|align=center|6
 +
|align=center|4
 +
|align=center|7
|-
|-
-
|align="center"|Positive control
+
|style="background: #ccccff;"| L127
-
|align="center"|155 (transformation efficiency:1.5*10^7/ug)
+
|align=center| D125
-
|align="center"|???
+
|align=center| D119
-
|align="center"|
+
|align=center| A
 +
|align=center|6
 +
|align=center|3
 +
|align=center|8
|}
|}

Latest revision as of 17:04, 13 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →


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
Retrieved from "http://2008.igem.org/Team:Paris/July_31"