Team:Paris/August 23

From 2008.igem.org

(Difference between revisions)
(→Construction of pFlgA - YFP tripart (+/- LVA))
(→Digestion)
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|18.04
|18.04
|XbaI and PstI
|XbaI and PstI
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|}
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=Promoter characterization plasmids a modifier=
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 +
==Transformation of ligations from August 20th==
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Top 10 cells were used
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[[Team:Paris/Notebook/Protocols#Transformation |Protocol]]
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 +
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{|border="1" style="text-align: center"
 +
|'''Ligation name'''
 +
|'''Vector digestion'''
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|'''Vector description'''
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|'''Vector conc. ug/mL'''
 +
|'''Vector volume'''
 +
|'''Insert digestion'''
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|'''Insert description'''
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|'''Insert conc. ug/mL'''
 +
|'''Insert volume'''
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|'''Product description'''
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|'''Antibiotic'''
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|-
 +
|L155
 +
|D164
 +
|J23101 promoter
 +
|3
 +
|16
 +
|D163
 +
|gfp generator
 +
|14
 +
|8
 +
|J23101 promoter-gfp generator
 +
|Amp
 +
|-
 +
|L156
 +
|D161
 +
|pTet promoter
 +
|39
 +
|1.25
 +
|D163
 +
|gfp generator
 +
|14
 +
|5
 +
|pTet promoter-gfp generator
 +
|Kana
 +
|-
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|Control L156
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|D161
 +
|
 +
|
 +
|1.25
 +
|none
 +
|
 +
|
 +
|
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|Vector autoligation control
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|Kana
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|-
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|L157
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|D125.2
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|B0015
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|15
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|3
 +
|D162
 +
|tetR
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|11
 +
|4
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|tetR-B0015
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|Kana
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|-
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|Control L157
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|D125.2
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|
 +
|
 +
|3
 +
|none
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|
 +
|
 +
|
 +
|Vector autoligation control
 +
|Kana
 +
|-
 +
|L166
 +
|D185
 +
|RBS B0032
 +
|21
 +
|2
 +
|D182
 +
|tetR
 +
|10
 +
|6.5
 +
|RBS B0032 - tetR
 +
|Amp
 +
|-
 +
|Control L166
 +
|D185
 +
|
 +
|
 +
|2
 +
|none
 +
|
 +
|
 +
|
 +
|Vector autoligation control
 +
|Amp
 +
|-
 +
|L167
 +
|D181
 +
|pTet
 +
|1
 +
|14
 +
|D184
 +
|gfp generator
 +
|14
 +
|3
 +
|gfp generator - pTet
 +
|Amp
 +
|-
 +
|Control L167
 +
|D181
 +
|pTet
 +
|
 +
|14
 +
|none
 +
|
 +
|
 +
|
 +
|Vector autoligation control
 +
|Amp
|}
|}

Revision as of 16:48, 26 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →


Contents

Construction for FIFO

Aim : Construction of pFlgA - YFP tripart (+/- LVA) "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png


Results of the transformation we did Yesterday

Ligation name Description Antibio Number Colonies observed Fluorescence Comments
Ligations
L160 D166(FV) - D132(FI)
pFlgA - YFP tripart (LVA-) 		Amp 44 No ok
L161 D167(FV) - D132(FI)
pFlgA - YFP tripart (LVA+) 		Amp 3 No ok
Controls
TL160 D166 (FV)
YFP tripart (LVA-) 		Amp (+++) No -
TL161 D167 (FV)
YFP tripart (LVA+) 		Amp 0 No ok
Positive Control pUC19 Amp 720 (efficiency 7.2.10^7) No OK 
=> Need to screen to know which clones we can use for the  of  pFlgA promotor cconstruction.

Construction of pFlgA - GFP Generator

Aim : Construction of "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

Transformation of the ligations we did yesterday

Protocol

Ligation Name Description Antibio
L164 D168 (FV) + D132 (FI)
pFlgA - gfp generator 		Amp
Control L164 D168
gfp generator 		Amp

Construction for Synchronization

Results of the transformation we did yesterday

Number of colonies

Ligation name Description Antibio Number Colonies observed Fluorescence Comments
Ligations
L158 D110(BV) - D131(BI)
rbs-TetR - gfp tripart 		Amp - - to do again
L159 D125(FV) - D109(FI)
rbs-LasI - double terminator 		Kan - - to do again
L162 D107(BV) - D163(BI)
rbs-LasI - gfp generator 		Amp - - to do again
L163 D110(BV) - D163(BI)
rbs-TetR - gfp generator 		Amp - - to do again
Controls
TL158 D110(BV) Amp - - to do again
TL159 D125(FV) Kan - - to do again
TL162 D107(BV) Amp - - to do again
TL163 D110(BV) Amp - - to do again
Positive Control pUC19 Amp 358 (efficiency 3.5.10^7) No OK 
=> We decided to do again the digestions of the ligation's reactions

Digestion

Protocol

Name Template DNA Description Vol MP (µl) Vol H2O (µl) Enzymes
D107 MP105.1 rbs-LasI (BV) 14.7 (0.5µg of plasmid) 11 SpeI / PstI
D109 MP105.1 rbs-LasI (FI) 14.7 11 EcoRI / SpeI
D110 MP106 rbs-TetR (BV) 9 15.7 SpeI / PstI
D125 MP118.1 Double terminator (FV) 4.54 20.16 EcoRI and XbaI
D163 MP143 gfp generator (BI) 6.66 18.04 XbaI and PstI


Promoter characterization plasmids a modifier

Transformation of ligations from August 20th

Top 10 cells were used


Protocol


Ligation name Vector digestion Vector description Vector conc. ug/mL Vector volume Insert digestion Insert description Insert conc. ug/mL Insert volume Product description Antibiotic
L155 D164 J23101 promoter 3 16 D163 gfp generator 14 8 J23101 promoter-gfp generator Amp
L156 D161 pTet promoter 39 1.25 D163 gfp generator 14 5 pTet promoter-gfp generator Kana
Control L156 D161 1.25 none Vector autoligation control Kana
L157 D125.2 B0015 15 3 D162 tetR 11 4 tetR-B0015 Kana
Control L157 D125.2 3 none Vector autoligation control Kana
L166 D185 RBS B0032 21 2 D182 tetR 10 6.5 RBS B0032 - tetR Amp
Control L166 D185 2 none Vector autoligation control Amp
L167 D181 pTet 1 14 D184 gfp generator 14 3 gfp generator - pTet Amp
Control L167 D181 pTet 14 none Vector autoligation control Amp
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