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Cloning of FlhB promoter
Protocol
- Preparation of the template :
Resuspension of 1 colony E.coli K12 strain MG 1655 in 100µl of water.
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
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| 10
| 11
| 12
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| Sample
| 100 bp ladder
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| Expected size (pb)
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| Measured size (pb)
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Construction for FIFO
Aim : Construction of pFlgA - YFP tripart (+/- LVA) "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)    
Digestion
Digestion
Protocol Digestion
| Name
| Template DNA
| Description
| Vol MP (µl)
| Vol H2O (µl)
| Enzymes
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| D166
| MP165.1
| RBS+ YFP LVA- term - FV
| 7.63
| 17
| EcoRI and XbaI
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| D167
| MP166.1
| RBS+ YFP LVA+ term - FV
| 8.9
| 15.8
| EcoRI and XbaI
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Protocol
 Gel Extraction of D166-D167
| Well
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
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| Sample
| 1kb ladder
| MP165.1
| MP166.1
| no sample
| D166
| no sample
| D167
| no sample
| 100pb ladder
| no sample
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| Expected size (pb)
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| 2 957
| 2 996
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| 2942
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| 2981
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| Measured size (pb)
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| 3 000
| 3 000
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Measurement of the concentration of D166 & D167 purified
Protocol (it's same that for Miniprep)
| Digestion Name
| Concentration (µg/mL)
| Ratio 260/280
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| D166
| 11
| 4.73
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| D167
| 10
| 2.84
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Ligation
Protocol
| Ligation Name
| Vector Name
| Volume Vector (µL)
| Insert
| Volume Insert (µL)
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| L160
| D166
| 3.63
| D132
| 2.03
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| L161
| D167
| 4.00
| D132
| 2.01
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| Control L160
| D166
| 3.63
| -
| -
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| Control L161
| D167
| 4.00
| -
| -
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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)
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| well n°
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| 15
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| 17
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| sample
| 1 kb DNA ladder
| positive control
| negative control
| S159.1
| 100 bp DNA ladder
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| colonie n°
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| 2
| 3
| 4
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| 8
| 9
| 10
| 11
| 12
| 13
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| expected size
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| 1192 bp
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| measured size
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| 1,5 kb
1,1 kb
0,6 kb
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| Gel n°2 (FlhDC+promotor in pSB1A2)
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| well n°
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| 3
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| 7
| 8
| 9
| 10
| 11
| 12
| 13
| 14
| 15
| 16
| 17
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| sample
| 1 kb DNA ladder
| positive control
| negative control
| S161.1
| 100 bp DNA ladder
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| colonie n°
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| 1
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| 4
| 5
| 6
| 7
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| 10
| 11
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| 13
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| expected size
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| 1403 bp
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| measured size
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| 0,3 kb
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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)
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| L158
| rbs-TetR + gfp tripart
 
| D110 (BV)
| 2
| D131 (BI)
| 2.89
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| L159
| rbs-lasI + Double terminator
| D125 (FV)
| 2.08
| D109 (FI)
| 1.15
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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
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| D179
| MP3.4
| B0015 (double terminator B0010-B0012) - BV
| 4
| SpeI and PstI
| 9
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| D180
| MP101.1
| promoter J23101- BV
| 1
| SpeI and PstI
| 7
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| D181
| MP104.2
| PTet (TetR repressible promoter) - FV
| 1
| EcoRI and XbaI
| 1
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| D182
| MP114.1
| TetR - BI
| 1
| XbaI and PstI
| 10
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| D183
| MP119.3
| pBad promoter - BI
| 1
| XbaI and PstI
| 0
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| D184
| MP143.1
| gfp generator - FI
| 2
| EcoRI and SpeI
| 13
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| D185
| MP163.1
| B0032 RBS - BV
| 2
| SpeI and PstI
| 21
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D179
D180
D181
D182
D183
D184
D185
Ligation
Protocol
| Ligation name
| Vector digestion
| Vector description
| Vector volume
| Insert digestion
| Insert description
| Insert volume
| Product description
| Antibiotic
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| L155
| D164
| J23101 promoter
| 10
| D163
| gfp generator
| 2
| J23101 promoter-gfp generator
| Amp
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| L156
| D161
| pTet promoter
| 1
| D163
| gfp generator
| 4
| pTet promoter-gfp generator
| Kana
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| D161
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| 1
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| Vector autoligation control
| Kana
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| L157
| D125.2
| B0015
| 3
| D162
| 4
| tetR
| tetR-B0015
| Amp
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| D125.2
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| 3
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| Vector autoligation control
| Amp
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