Team:Paris/August 1

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minipreps
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{{Paris/Calendar_Links|July 31|August 2}}
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transformation
 
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souche stockage
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== MiniPreps ==
 +
 
 +
=== Conditions ===
 +
 
 +
* Use of Quiagen's protocol on all the clones cultivated on the 31th.
 +
* Preparation of 50µl of minipreps in water.
 +
 
 +
 
 +
=== List of Minipreps ===
 +
 
 +
{| border="1"
 +
|align="center"|'''Name'''
 +
|align="center"|'''Biobricks'''
 +
|align="center"|'''Description'''
 +
|-
 +
|align="center"|MP124
 +
|align="center"|Strong rbs - YFP<br>D129 (BV) -  D118 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|MP125
 +
|align="center"|Strong rbs - mRFP<br>D129 (BV) -  D122 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|MP126 
 +
|align="center"|Strong rbs - lasR activator<br>D129 (BV) -  D114 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]
 +
|-
 +
|align="center"|MP127
 +
|align="center"|Strong promoter - ECFP<br>D123 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP128
 +
|align="center"|Strong promoter - gfp Tripart<br>D123 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP129
 +
|align="center"|Strongest promoter - ECFP<br>D103 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP130
 +
|align="center"|Strong promoter -  gfp Tripart<br>D103 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP131
 +
|align="center"|Medium promoter - gfp Tripart<br>D124 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP132
 +
|align="center"|Weak promoter - ECFP<br>D104 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP133
 +
|align="center"|Weak promoter - gfp<br>D104 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|MP134
 +
|align="center"|pLas - ECFP<br>D105 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP135
 +
|align="center"|pLas - gfp Tripart<br>D105 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP136
 +
|align="center"|yfp - Double Terminator<br>D125 (FV) - D117 (FI)
 +
|align="center"|[[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP137
 +
|align="center"|rfp - Double Terminator<br>D125 (FV) - D121 (FI)
 +
|align="center"|[[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP138
 +
|align="center"|lasR activator + LVA - Double Terminator<br>D125 (FV) - D113 (FI)
 +
|align="center"|[[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP139
 +
|align="center"|Strong promoter - gfp tripart<br>D106 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|MP140
 +
|align="center"|Strongest RBS - rfp<br>D102 (BV) - D122 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|MP141
 +
|align="center"|Strongest RBS - yfp<br>D102 (BV) - D118 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]]
 +
|}
 +
 
 +
== Glycerol Stock ==
 +
 
 +
=== Protocol ===
 +
* 1mL glycerol
 +
* 1mL culture in LB
 +
 
 +
 
 +
=== List of Strain stocked ===
 +
 
 +
{|border="1"
 +
|align="center"|'''Strain'''
 +
|align="center"|'''Ligation'''
 +
|align="center"|'''Biobricks'''
 +
|align="center"|'''Description'''
 +
|- 
 +
|align="center"|S123
 +
|align="center"|L102
 +
|align="center"|Strong rbs - YFP<br>D129 (BV) -  D118 (BI) 
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|S124
 +
|align="center"|L103
 +
|align="center"|Strong rbs - mRFP<br>D129 (BV) - D122 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|S125
 +
|align="center"|L104
 +
|align="center"|Strong rbs - lasR activator<br>D129 (BV) - D114 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]
 +
|-
 +
|align="center"|S126
 +
|align="center"|L105
 +
|align="center"|Strong promoter - ECFP<br>D123 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|- 
 +
|align="center"|S127
 +
|align="center"|L106
 +
|align="center"|Strong promoter -  gfp Tripart<br>D123 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|- 
 +
|align="center"|S128
 +
|align="center"|L107
 +
|align="center"|Strongest promoter - gfp Tripart<br>D103 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S129
 +
|align="center"|L108
 +
|align="center"|Weak promoter - ECFP<br>D103 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S130
 +
|align="center"|L110
 +
|align="center"|Medium promoter - gfp<br>D124 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|S131
 +
|align="center"|L111
 +
|align="center"|Weak promoter - ECFP<br>D104 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S132
 +
|align="center"|L112
 +
|align="center"|Weak promoter - gfp Tripart<br>D104 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|- 
 +
|align="center"|S133
 +
|align="center"|L115
 +
|align="center"|pLas - ECFP <br>D105 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_reporter.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S134
 +
|align="center"|L116
 +
|align="center"|pLas - gfp Tripart<br>D105 (BV) - D131 (BI)
 +
|align="center"|[[Image:Part_icon_reporter.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S135
 +
|align="center"|L117
 +
|align="center"|yfp - Double Terminator<br>D125 (FV) - D117 (FI)
 +
|align="center"|[[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S136
 +
|align="center"|L118
 +
|align="center"|rfp - Double Terminator<br>D125 (FV) - D117 (FI)
 +
|align="center"|[[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S137
 +
|align="center"|L119
 +
|align="center"|LasR activator + LVA - Double terminator<br>D125 (FV) - D113 (FI)
 +
|align="center"|[[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S138
 +
|align="center"|L121
 +
|align="center"|Strong promoter - gfp Tripart<br>D106 (BV) - D130 (BI)
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|S139
 +
|align="center"|L124
 +
|align="center"|Strongest rbs - mRFP<br>D102 (BV) - D122 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|S140
 +
|align="center"|L125
 +
|align="center"|Strongest rbs - yfp<br>D102 (BV) - D118 (BI)
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]]
 +
|}
 +
 
 +
== Transformations ==
 +
 
 +
=== Protocol ===
 +
 
 +
''use of TOP10 Chemically competent cells''
 +
 
 +
* Defroze competent cells on ice during 5'
 +
* Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
 +
* Incubate 30' on ice
 +
* Heat-shock the cells during 30" at 42°C without shaking
 +
* Put 2' on ice
 +
* Add 250µL of pre-warmed SOC medium (4°C)
 +
* Incubate 1h at 37°C under shaking (225rpm)
 +
* Spin at 5.000rpm during 30"
 +
* Remove 150µL of supernatant
 +
* Resuspent the pellet in the 150µL left
 +
* Spread on adequated plates
 +
* Incubate O/N at 37°C
 +
 
 +
 
 +
=== List of transformation ===
 +
 
 +
{| border="1"
 +
|align="center"|'''Name'''
 +
|align="center"|'''Description'''
 +
|align="center"|'''Biobrick'''
 +
|-
 +
|align="center"|L100
 +
|align="center"| RBS-TetR-ECFP
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|L101
 +
|align="center"|RBS-tetR-GFP tripart
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|L113
 +
|align="center"|pBAD-ECFP
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|L114
 +
|align="center"|pBAD-GFP tripart
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_reporter.png]]
 +
|-
 +
|align="center"|L120
 +
|align="center"|ptet-ECFP
 +
|align="center"|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|L122
 +
|align="center"|RBS-lasI-ECFP
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|L123
 +
|align="center"|RBS-lasI-GFP-ter
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]]
 +
|-
 +
|align="center"|L126
 +
|align="center"|RBS-lasR-LVA
 +
|align="center"|[[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]
 +
|}

Latest revision as of 16:35, 23 August 2008

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Contents

MiniPreps

Conditions

  • Use of Quiagen's protocol on all the clones cultivated on the 31th.
  • Preparation of 50µl of minipreps in water.


List of Minipreps

Name Biobricks Description
MP124 Strong rbs - YFP
D129 (BV) - D118 (BI) 		Part icon rbs.pngPart icon reporter.png
MP125 Strong rbs - mRFP
D129 (BV) - D122 (BI) 		Part icon rbs.pngPart icon reporter.png
MP126 Strong rbs - lasR activator
D129 (BV) - D114 (BI) 		Part icon rbs.pngIcon coding.png
MP127 Strong promoter - ECFP
D123 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP128 Strong promoter - gfp Tripart
D123 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP129 Strongest promoter - ECFP
D103 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP130 Strong promoter - gfp Tripart
D103 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP131 Medium promoter - gfp Tripart
D124 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP132 Weak promoter - ECFP
D104 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP133 Weak promoter - gfp
D104 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon reporter.png
MP134 pLas - ECFP
D105 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP135 pLas - gfp Tripart
D105 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP136 yfp - Double Terminator
D125 (FV) - D117 (FI) 		Part icon reporter.pngPart icon terminator.pngPart icon terminator.png
MP137 rfp - Double Terminator
D125 (FV) - D121 (FI) 		Part icon reporter.pngPart icon terminator.pngPart icon terminator.png
MP138 lasR activator + LVA - Double Terminator
D125 (FV) - D113 (FI) 		Icon coding.pngPart icon terminator.pngPart icon terminator.png
MP139 Strong promoter - gfp tripart
D106 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
MP140 Strongest RBS - rfp
D102 (BV) - D122 (BI) 		Part icon rbs.pngPart icon reporter.png
MP141 Strongest RBS - yfp
D102 (BV) - D118 (BI) 		Part icon rbs.pngPart icon reporter.png

Glycerol Stock

Protocol

  • 1mL glycerol
  • 1mL culture in LB


List of Strain stocked

Strain Ligation Biobricks Description
S123 L102 Strong rbs - YFP
D129 (BV) - D118 (BI) 		Part icon rbs.pngPart icon reporter.png
S124 L103 Strong rbs - mRFP
D129 (BV) - D122 (BI) 		Part icon rbs.pngPart icon reporter.png
S125 L104 Strong rbs - lasR activator
D129 (BV) - D114 (BI) 		Part icon rbs.pngIcon coding.png
S126 L105 Strong promoter - ECFP
D123 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S127 L106 Strong promoter - gfp Tripart
D123 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S128 L107 Strongest promoter - gfp Tripart
D103 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S129 L108 Weak promoter - ECFP
D103 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S130 L110 Medium promoter - gfp
D124 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon reporter.png
S131 L111 Weak promoter - ECFP
D104 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S132 L112 Weak promoter - gfp Tripart
D104 (BV) - D131 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S133 L115 pLas - ECFP
D105 (BV) - D130 (BI) 		Part icon reporter.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S134 L116 pLas - gfp Tripart
D105 (BV) - D131 (BI) 		Part icon reporter.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S135 L117 yfp - Double Terminator
D125 (FV) - D117 (FI) 		Part icon reporter.pngPart icon terminator.pngPart icon terminator.png
S136 L118 rfp - Double Terminator
D125 (FV) - D117 (FI) 		Part icon reporter.pngPart icon terminator.pngPart icon terminator.png
S137 L119 LasR activator + LVA - Double terminator
D125 (FV) - D113 (FI) 		Icon coding.pngPart icon terminator.pngPart icon terminator.png
S138 L121 Strong promoter - gfp Tripart
D106 (BV) - D130 (BI) 		Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
S139 L124 Strongest rbs - mRFP
D102 (BV) - D122 (BI) 		Part icon rbs.pngPart icon reporter.png
S140 L125 Strongest rbs - yfp
D102 (BV) - D118 (BI) 		Part icon rbs.pngPart icon reporter.png

Transformations

Protocol

use of TOP10 Chemically competent cells

  • Defroze competent cells on ice during 5'
  • Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
  • Incubate 30' on ice
  • Heat-shock the cells during 30" at 42°C without shaking
  • Put 2' on ice
  • Add 250µL of pre-warmed SOC medium (4°C)
  • Incubate 1h at 37°C under shaking (225rpm)
  • Spin at 5.000rpm during 30"
  • Remove 150µL of supernatant
  • Resuspent the pellet in the 150µL left
  • Spread on adequated plates
  • Incubate O/N at 37°C


List of transformation

Name Description Biobrick
L100 RBS-TetR-ECFP Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
L101 RBS-tetR-GFP tripart Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
L113 pBAD-ECFP Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
L114 pBAD-GFP tripart Part icon regulatory.pngPart icon reporter.png
L120 ptet-ECFP Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
L122 RBS-lasI-ECFP Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.png
L123 RBS-lasI-GFP-ter Part icon rbs.pngIcon coding.pngPart icon reporter.pngPart icon terminator.png
L126 RBS-lasR-LVA Part icon rbs.pngIcon coding.png
Retrieved from "http://2008.igem.org/Team:Paris/August_1"