Team:Paris/August 23

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Construction of pFlgA - YFP tripart (+/- LVA)

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


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) Amp (+++) No -
TL161 D167 (FV) 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
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