Team:Paris/August 21

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==Ligation==
==Ligation==

Revision as of 16:51, 26 August 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

[[Image:KR00019b.jpg| thumb| 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

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 single colonies with the dilution 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 positive control negative 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 positive control negative 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

Ligation

Our ligations from yesterday didn't work. The positive control for transformation worked.

Digestion

We had a problem with a gel extraction so we have to make again the digestions from yesterday


Other digestions made:


Protocol Digestion

Digestion name Plasmid Description Miniprep used Enzymes Concentration after gel extraction
D179 MP3.4 B0015 (double terminator B0010-B0012) - BV 4 SpeI and PstI 9
D180 MP101.1 promoter J23101- BV 1 SpeI and PstI 7
D181 MP104.2 PTet (TetR repressible promoter) - FV 1 EcoRI and XbaI 1
D182 MP114.1 TetR - BI 1 XbaI and PstI 10
D183 MP119.3 pBad promoter - BI 1 XbaI and PstI 0
D184 MP143.1 gfp generator - FI 2 EcoRI and SpeI 13
D185 MP163.1 B0032 RBS - BV 2 SpeI and PstI 21


D179 D180 D181 D182 D183 20-08-08.png 20-08-08-coupe.png

D184 D185 20-08-08bis.png 20-08-08bis-coupe.png


Ligation

Protocol


Ligation name Vector digestion Vector description Vector volume Insert digestion Insert description Insert volume Product description Antibiotic
L155 D164 J23101 promoter 10 D163 gfp generator 2 J23101 promoter-gfp generator Amp
L156 D161 pTet promoter 1 D163 gfp generator 4 pTet promoter-gfp generator Kana
D161 1 Vector autoligation control Kana
L157 D125.2 B0015 3 D162 4 tetR tetR-B0015 Amp
D125.2 3 Vector autoligation control Amp
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