Team:Paris/August 26
From 2008.igem.org
(→Minipreps and glycerol stock) |
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=Extraction of EnvZ* and OmpR* from ''E. coli'' genome= | =Extraction of EnvZ* and OmpR* from ''E. coli'' genome= | ||
==Name of the PCR== | ==Name of the PCR== | ||
- | {|border="2" | + | {|style="text-align: center;" border="2" |
|Name | |Name | ||
|What's in ? | |What's in ? | ||
Line 34: | Line 34: | ||
|O 139 | |O 139 | ||
|} | |} | ||
+ | |||
==Protocol== | ==Protocol== | ||
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|- | |- | ||
|style="background: #cbff7B"|T 147 | |style="background: #cbff7B"|T 147 | ||
- | | | + | |no sample |
|3 | |3 | ||
- | | | + | |colspan="2"| |
- | | | + | |
|- | |- | ||
|style="background: #cbff7B"|PCR 148 | |style="background: #cbff7B"|PCR 148 | ||
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|- | |- | ||
|style="background: #cbff7B"|T 148 | |style="background: #cbff7B"|T 148 | ||
- | | | + | |no sample |
|5 | |5 | ||
- | | | + | |colspan="2"| |
- | | | + | |
|} | |} | ||
Conclusion : All the PCR worked perfectly well ! | Conclusion : All the PCR worked perfectly well ! | ||
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==PCR Promoters FlhDC and Gene== | ==PCR Promoters FlhDC and Gene== | ||
- | * pFlhDC (O111-F / O113-R) | + | * PCR 137 = pFlhDC (O111-F / O113-R) |
- | * Gene FlhDC (O134-F / O131-R) | + | * PCR 141 = Gene FlhDC (O134-F / O131-R) |
- | * pFlgB (O102-F / O103-R) | + | * PCR 125 = pFlgB (O102-F / O103-R) |
- | * pFlhB (O108-F / O109-R) | + | * PCR 126 = pFlhB (O108-F / O109-R) |
---- | ---- | ||
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==PCR mutagenesis FliA== | ==PCR mutagenesis FliA== | ||
- | * PCRFliA1 (O143-F / O152-R) | + | * PCRFliA1 (O143-F / O152-R) |
- | * PCRFliA2 (O153-F / O150-R) | + | * PCR 145 = PCRFliA1' (O148-F / O152-R) |
+ | * PCR 146 = PCRFliA2 (O153-F / O150-R) | ||
* PCRFliA3 (O148-F / O150-R) | * PCRFliA3 (O148-F / O150-R) | ||
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|style="background: #cbff7B"| ~ 1000 bp | |style="background: #cbff7B"| ~ 1000 bp | ||
|} | |} | ||
+ | |||
=Cloning of EnvZ*= | =Cloning of EnvZ*= | ||
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{|border="1" style="text-align:center" | {|border="1" style="text-align:center" | ||
- | |||
|'''Digestion n°''' | |'''Digestion n°''' | ||
+ | |'''Name''' | ||
|'''[DNA] in µg/mL''' | |'''[DNA] in µg/mL''' | ||
|'''A260/A280''' | |'''A260/A280''' | ||
|- | |- | ||
- | |||
|D159 | |D159 | ||
+ | |EnvZ* | ||
|10 µg/mL | |10 µg/mL | ||
|1.35 | |1.35 | ||
|- | |- | ||
- | |||
|D116 | |D116 | ||
+ | |pSB1A2 | ||
|17 µg/mL | |17 µg/mL | ||
|1.02 | |1.02 | ||
Line 350: | Line 351: | ||
*transformation of TOP10 competent cells with 5 µL of ligation product | *transformation of TOP10 competent cells with 5 µL of ligation product | ||
*spreading on LB plates + ampicilline and incubation overnight at 37°C | *spreading on LB plates + ampicilline and incubation overnight at 37°C | ||
+ | |||
+ | |||
='''Miniprep and stock glycerolof New Biobrick'''= | ='''Miniprep and stock glycerolof New Biobrick'''= | ||
Line 355: | Line 358: | ||
*[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]] | *[[Team:Paris/Notebook/Protocols#Glycerol stocks| Protocol stocks]] | ||
*[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol miniprep]] | *[[Team:Paris/Notebook/Protocols#Minipreps (Kit_Qiagen)| Protocol miniprep]] | ||
- | |||
- | |||
{|border="1" style="text-align: center" | {|border="1" style="text-align: center" | ||
|'''Miniprep''' | |'''Miniprep''' | ||
|'''Strain''' | |'''Strain''' | ||
+ | |'''Antiobiotic''' | ||
|'''Name''' | |'''Name''' | ||
|'''Description''' | |'''Description''' | ||
|- | |- | ||
- | | | + | |MP101.1 |
- | | | + | |S109.1 |
- | |rowspan="2"| | + | |rowspan="2"|Amp |
- | |rowspan="2"| [[Image: | + | |rowspan="2"| J23101 |
+ | |rowspan="2"| [[Image:Part_icon_regulatory.png]]<br>constitutive promotor | ||
|- | |- | ||
- | | | + | |MP101.2 |
- | | | + | |S109.2 |
|- | |- | ||
- | + | |MP101.1 | |
- | | | + | |S109.1 |
- | | | + | |rowspan="2"|Amp |
- | |rowspan="2"| | + | |rowspan="2"| J23101 |
- | |rowspan="2"| [[Image: | + | |rowspan="2"| [[Image:Part_icon_regulatory.png]]<br>constitutive promotor |
|- | |- | ||
- | | | + | |MP101.2 |
- | | | + | |S109.2 |
|- | |- | ||
- | | | + | |MP104.1 |
- | | | + | |S111.1 |
- | |rowspan="2"| | + | |rowspan="2"|Amp |
- | |rowspan="2"| [[Image: | + | |rowspan="2"| R0040 |
+ | |rowspan="2"| [[Image:Part_icon_regulatory.png]]<br>ptetR repressible | ||
|- | |- | ||
- | | | + | |MP104.2 |
- | | | + | |S111.2 |
|- | |- | ||
- | | | + | |MP104.1 |
- | | | + | |S111.1 |
- | |rowspan="2"| | + | |rowspan="2"|Amp |
- | |rowspan="2"| [[Image: | + | |rowspan="2"| R0040 |
+ | |rowspan="2"| [[Image:Part_icon_regulatory.png]]<br>ptetR repressible | ||
|- | |- | ||
- | | | + | |MP104.2 |
- | | | + | |S111.2 |
|- | |- | ||
- | | | + | |MP105.1 |
- | | | + | |S112.1 |
- | |rowspan="2"| | + | |rowspan="2"|Amp |
- | |rowspan="2"| [[Image:Icon_rbs.png]][[Image:Icon_reporter | + | |rowspan="2"| S03154 |
+ | |rowspan="2"| [[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>RBS-lasI | ||
|- | |- | ||
- | | | + | |MP105.2 |
- | | | + | |S112.2 |
+ | |- | ||
+ | |MP105.1 | ||
+ | |S112.1 | ||
+ | |rowspan="2"|Amp | ||
+ | |rowspan="2"| S03154 | ||
+ | |rowspan="2"| [[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>RBS-lasI | ||
+ | |- | ||
+ | |MP105.2 | ||
+ | |S112.2 | ||
+ | |- | ||
+ | |MP143 | ||
+ | |S157 | ||
+ | |Amp | ||
+ | |E0240 (in pSB1A2) | ||
+ | |[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>RBS GFP dbl term | ||
+ | |- | ||
+ | |MP1151 | ||
+ | |S150 | ||
+ | |Amp | ||
+ | |L122 <br>D107 (BV) - D130 (BI) | ||
+ | |[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>RBS-lasI - ECFP | ||
+ | |- | ||
+ | |MP157 | ||
+ | |S156 | ||
+ | |Kan | ||
+ | |L138 (E0240 in pSB3K3) | ||
+ | |[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>gfp generator | ||
+ | |- | ||
+ | |MP158.1 | ||
+ | |S160.1 | ||
+ | |rowspan="3"|Amp | ||
+ | |rowspan="3"| L141 | ||
+ | |rowspan="3"| [[Image:Part_icon_regulatory.png]]<br>pFlhD | ||
+ | |- | ||
+ | |MP158.2 | ||
+ | |S160.2 | ||
+ | |- | ||
+ | |MP158.3 | ||
+ | |S160.3 | ||
+ | |- | ||
+ | |MP171 | ||
+ | |S171 | ||
+ | |Amp | ||
+ | |L167 <br>D181 (BV) - D184 (BI) | ||
+ | |[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_regulatory.png]]<br>gfp generator - pTet | ||
|} | |} | ||
+ | |||
+ | |||
='''Construction of pFlhB - mRFP Tripart (LVA+)'''= | ='''Construction of pFlhB - mRFP Tripart (LVA+)'''= | ||
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|colspan="2"| | |colspan="2"| | ||
|} | |} | ||
+ | |||
='''Screening of the cloning of pFlgA-GFP Generator'''= | ='''Screening of the cloning of pFlgA-GFP Generator'''= | ||
Line 525: | Line 579: | ||
|Amp | |Amp | ||
|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | |[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | ||
- | + | FlgA-GFP generator | |
|- | |- | ||
|MP175.2 | |MP175.2 | ||
Line 532: | Line 586: | ||
|Amp | |Amp | ||
|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | |[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | ||
- | + | FlgA-GFP generator | |
|- | |- | ||
|MP175.4 | |MP175.4 | ||
Line 539: | Line 593: | ||
|Amp | |Amp | ||
|[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | |[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]] | ||
- | <br> | + | FlgA-GFP generator |
+ | |} | ||
+ | |||
+ | |||
+ | ='''Construction for Synchronisation'''= | ||
+ | =='''Results of the transformation we did [[Team:Paris/August 24 |yesterday]]'''== | ||
+ | |||
+ | {|border="1" style="text-align: center" | ||
+ | |'''Ligation name''' | ||
+ | |'''Description''' | ||
+ | |'''Antibio''' | ||
+ | |'''Number Colonies observed''' | ||
+ | |'''Fluorescence''' | ||
+ | |'''Comments''' | ||
+ | |- | ||
+ | |colspan="6" |Ligations | ||
+ | |- | ||
+ | |L159 | ||
+ | |D109(FI) - D125(FV)<br>rbs-lasI - double terminator | ||
+ | |Kan | ||
+ | | - | ||
+ | | - | ||
+ | | to do again | ||
+ | |- | ||
+ | |L162 | ||
+ | |D107(BV) - D163(BI)<br>rbs-lasI - gfp generator | ||
+ | |Amp | ||
+ | | - | ||
+ | | - | ||
+ | | to do again | ||
+ | |- | ||
+ | |L162 | ||
+ | |D110(BV) - D163(BI)<br>rbs-TetR - gfp generator | ||
+ | |Amp | ||
+ | | - | ||
+ | | - | ||
+ | | to do again | ||
+ | |- | ||
+ | |colspan="6" |Controls | ||
+ | |- | ||
+ | |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 | ||
+ | | | ||
+ | |No | ||
+ | |OK | ||
|} | |} |
Latest revision as of 17:59, 11 September 2008
Extraction of EnvZ* and OmpR* from E. coli genomeName of the PCR
ProtocolProtocol
PCR Programme
ResutsElectrophoresis settings
Results of the electrophoresis
Conclusion : All the PCR worked perfectly well ! Cleaning of the PCR productsThe cleaned PCR products are stored in the freezer overnight.
PCR Promoters and Genes FlhDC/FliAPCR Promoters FlhDC and Gene
Program: Gradient Vf=20µL PCR Promoters FliA
Program: promoter Vf=50µL PCR mutagenesis FliA
Program: PCRFliA1' Program: PCRFliA2 Program: PCRFliA3
Cloning of EnvZ*Concentration measurement by Biophotometer
Ligation
Miniprep and stock glycerolof New Biobrick
Construction of pFlhB - mRFP Tripart (LVA+)Aim : Construction of "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078)
DigestionDigestion
Gel Verification
Screening of the cloning of pFlgA-GFP GeneratorElectrophoresis
Minipreps and glycerol stock
Construction for SynchronisationResults of the transformation we did yesterday
|