From 2008.igem.org
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| | |gfp generator | | |gfp generator |
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| | |gfp generator - pTet | | |gfp generator - pTet |
| | |Amp | | |Amp |
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Revision as of 17:11, 26 August 2008
|
← Yesterday ↓ Calendar ↑Tomorrow →
Construction for FIFO
Aim : Construction of pFlgA - YFP tripart (+/- LVA) "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)    
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)
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 of ligations from August 20th
Top 10 cells were used
Protocol
Promoter characterization plasmids
Transformation of ligations from August 20th
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
| ++
| Control L166
| D185
|
| none
|
| Vector autoligation control
| Amp
| ++
| L167
| D181
| pTet
| D184
| gfp generator
| gfp generator - pTet
| Amp
| 1
|
|
"
