Team:Paris/August 23

From 2008.igem.org

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='''Construction of pFlgA - GFP Generator'''=
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='''Construction for Synchronization'''=
='''Construction for Synchronization'''=

Revision as of 14:46, 6 September 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

Transformation results: ligations from August 21th

Top 10 cells were used

Protocol

Ligation name Vector digestion Vector description Insert digestion Insert description Product description Antibiotic Number of colonies
L155 D164 J23101 promoter D163 gfp generator J23101 promoter-gfp generator Amp 0
L156 D161 pTet promoter D163 gfp generator pTet promoter-gfp generator Kana 0
Control L156 D161 none Vector autoligation control Kana 1
L157 D125.2 B0015 D162 tetR tetR-B0015 Kana 1
Control L157 D125.2 none Vector autoligation control Kana 0
L166 D185 RBS B0032 D182 tetR RBS B0032 - tetR Amp 68
Control L166 D185 none Vector autoligation control Amp 49
L167 D181 pTet D184 gfp generator gfp generator - pTet Amp 1
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