Team:Paris/August 21

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='''Construction for FIFO '''=
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=Construction for FIFO=
Aim : Construction of pFlgA - YFP tripart (+/- LVA) ''' "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
Aim : Construction of pFlgA - YFP tripart (+/- LVA) ''' "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]

Revision as of 14:12, 6 September 2008

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Contents

Cloning of FlhB promoter

Protocol

  • Preparation of the template :

Resuspension of 1 colony E.coli K12 strain MG 1655 in 100µl of water.


  • Preparation of PCR mix :

1µl of dNTP
10µl Buffer Phusion 5X
2.5µl O 108
2.5µl O 109
1µl Template
1µl Phusion
33µL pure water

Result

File:KR00019b.jpg
Vérification of pFlhB
Well 1 2 3 4 5 6 7 8 9 10 11 12
Sample 100 bp ladder
Expected size (pb)
Measured size (pb)

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

Digestion

Digestion

Protocol Digestion

Name Template DNA Description Vol MP (µl) Vol H2O (µl) Enzymes
D166 MP165.1 RBS+ YFP LVA- term - FV 7.63 17 EcoRI and XbaI
D167 MP166.1 RBS+ YFP LVA+ term - FV 8.9 15.8 EcoRI and XbaI

Gel Extraction

Protocol

Gel Extraction of D166-D167
Well 1 2 3 4 5 6 7 8 9 10 11 12
Sample 1kb
ladder 		MP165.1 MP166.1 no sample D166 no sample D167 no sample 100pb
ladder 		no sample
Expected size (pb) 2 957 2 996 2942 2981
Measured size (pb) 3 000 3 000 2 800 3 000

Measurement of the concentration of D166 & D167 purified

Protocol (it's same that for Miniprep)

Digestion Name Concentration (µg/mL) Ratio 260/280
D166 11 4.73
D167 10 2.84

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L160 D166 3.63 D132 2.03
L161 D167 4.00 D132 2.01
Control L160 D166 3.63 - -
Control L161 D167 4.00 - -

Screening of the cloning of E0240 and FlhDC+promotor

We obtained colonies isolated with the dilution 1/100; 13 clones were analysed by PCR

PCR screening

reaction mixture (25 µL)

  • 12,5 µL Quick load PCR Mixture 2X
  • 0,5 µL O18
  • 0,5 µL O19
  • 11,5 µL water

PCR screening programm

  • elongation time: 1 min 30
  • primers: O18 and O19
  • positive control: S158 (pSB3K3)
  • negative control: no template

Electrophoresis

Gel n°1 (E0240 screening)
Gel n°2 (FlhDC+promotor screening)
  • 1% agarose gel
  • 10 µL loaded
Gel n°1 (E0240 in pSB3K3)
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
sample 1 kb
DNA ladder 		control + control - S159.1 100 bp
DNA ladder
colonie n° 1 2 3 4 5 6 7 8 9 10 11 12 13
expected size 1192 bp
measured size 1,5 kb
1,1 kb
0,6 kb
Gel n°2 (FlhDC+promotor in pSB1A2)
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
sample 1 kb
DNA ladder 		control + control - S161.1 100 bp
DNA ladder
colonie n° 1 2 3 4 5 6 7 8 9 10 11 12 13
expected size 1403 bp
measured size 0,3 kb 


Results: 
*The clone of E0240 (S159.1) always have several bands amplified by PCR.
It might contain different plasmids.
*The clone of FlhDC+promotor (S161.1) don't have the correct size band. 
It also doesn't have the insert in the plasmid.

Construction for synchronization

Ligations

Protocol

Ligation Name Description Vector Name Volume vector (µL) Insert Name Volume insert(µL)
L158 rbs-TetR + gfp tripart
Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png 		D110 (BV) 2 D131 (BI) 2.89
L159 rbs-lasI + Double terminator
Part icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png 		D125 (FV) 2.08 D109 (FI) 1.15

Promoter characterization plasmids

Ligations from digestions from 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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