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Team:INSA Toulouse/contenu/lab practice/notebook/calendar/carry
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| - | <img src="https://static.igem.org/mediawiki/2013/ | + | <img src="https://static.igem.org/mediawiki/2013/6/68/Carry_carac.png" class="imgcontent" /> |
<div class="clear"></div> | <div class="clear"></div> | ||
| + | <div class="accordeon"> | ||
| - | < | + | <h2 class="faq" title="Click to open the FAQ">June 2013</h2> |
| - | + | <div class="infos"> | |
| - | + | ||
| - | < | + | |
<ul class="circlearrow"> | <ul class="circlearrow"> | ||
<li><span class="spantitle2">Week 3 (24-30 June)</span></br> | <li><span class="spantitle2">Week 3 (24-30 June)</span></br> | ||
| - | Amplification of new biobricks for cloning :<br> | + | Amplification of new biobricks for cloning:<br> |
BBa_K081008: rbs Lux I,<br> | BBa_K081008: rbs Lux I,<br> | ||
BBa_I0462: rbs Lux R term, <br> | BBa_I0462: rbs Lux R term, <br> | ||
| Line 120: | Line 171: | ||
BBa_K081014: rbs RFP term,<br> | BBa_K081014: rbs RFP term,<br> | ||
<br> | <br> | ||
| - | We tested a new method for midipreps using kits : alkaline lysis with spin columns. Results were really better in terms of samples purity. Cloning can start!!!</li> | + | We tested a new method for midipreps using kits: alkaline lysis with spin columns. Results were really better in terms of samples purity. Cloning can start!!!</li> |
| - | </ | + | </div> |
| - | </ | + | </div> |
| - | < | + | |
| + | <div class="accordeon"> | ||
| + | <h2 class="faq" title="Click to open the FAQ">July 2013</h2> | ||
| + | <div class="infos"> | ||
<ul class="circlearrow"> | <ul class="circlearrow"> | ||
<li><span class="spantitle2">Week 4 (1-7 July)</span> | <li><span class="spantitle2">Week 4 (1-7 July)</span> | ||
<br> | <br> | ||
| - | The first necessary construction for carry characterization is the following : | + | The first necessary construction for carry characterization is the following: |
<img src="https://static.igem.org/mediawiki/2013/3/33/Carry_characterisation.png" class="imgcontent" /> | <img src="https://static.igem.org/mediawiki/2013/3/33/Carry_characterisation.png" class="imgcontent" /> | ||
Planning of cloning and study of the available methods for biobricks assembly. | Planning of cloning and study of the available methods for biobricks assembly. | ||
<br><br> | <br><br> | ||
| - | Assembly of first biobricks thanks to the iGEM 3A Assembly method :<br> | + | Assembly of first biobricks thanks to the iGEM 3A Assembly method:<br> |
- strong prom/rbs + Lux I (698 bp) in PSB1K3<br> | - strong prom/rbs + Lux I (698 bp) in PSB1K3<br> | ||
- pLux + rbs RFP term (936 bp) in PSB1A2<br> | - pLux + rbs RFP term (936 bp) in PSB1A2<br> | ||
| Line 142: | Line 196: | ||
<img src="https://static.igem.org/mediawiki/2013/a/a0/Prom_rbs_luxI_term.png" class="imgcontent" /> | <img src="https://static.igem.org/mediawiki/2013/a/a0/Prom_rbs_luxI_term.png" class="imgcontent" /> | ||
<img src="https://static.igem.org/mediawiki/2013/f/ff/LuxR_RFP.png" class="imgcontent" /><br> | <img src="https://static.igem.org/mediawiki/2013/f/ff/LuxR_RFP.png" class="imgcontent" /><br> | ||
| - | New round of 3A cloning : <br> | + | New round of 3A cloning: <br> |
- strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3<br> | - strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3<br> | ||
- strong prom/rbs + rbs Lux R term (991 bp) in PSB1K3<br><br> | - strong prom/rbs + rbs Lux R term (991 bp) in PSB1K3<br><br> | ||
| Line 148: | Line 202: | ||
<li><span class="spantitle2">Week 5 (8-14 July)</span><br> | <li><span class="spantitle2">Week 5 (8-14 July)</span><br> | ||
2 mL liquid cultures of 6 clones for each cloning and then minipreps. <br> | 2 mL liquid cultures of 6 clones for each cloning and then minipreps. <br> | ||
| - | 0,8% gel was run to confirm | + | 0,8% gel was run to confirm cloning success. <br> |
- correct insert for strong promoter strong rbs + Lux R terminator <br> | - correct insert for strong promoter strong rbs + Lux R terminator <br> | ||
- cloning failed for strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3<br> | - cloning failed for strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3<br> | ||
<br> | <br> | ||
New try for cloning of 08/08.<br> | New try for cloning of 08/08.<br> | ||
| - | Two new assembly with 3A method : <br> | + | Two new assembly with 3A method: <br> |
- strong promoter/rbs Lux R + pLux RFP in PSB1C3<br> | - strong promoter/rbs Lux R + pLux RFP in PSB1C3<br> | ||
- rbs cI term + pLux RFP in PSB1K3<br> | - rbs cI term + pLux RFP in PSB1K3<br> | ||
<br> | <br> | ||
2 mL liquid cultures of 6 clones for cI pLux RFP cloning and then minipreps. <br> | 2 mL liquid cultures of 6 clones for cI pLux RFP cloning and then minipreps. <br> | ||
| - | Bacterial carpets were obtained for the two other clonings : transformation with the remaining 5 µL ligation of 09/07.<br> | + | Bacterial carpets were obtained for the two other clonings: transformation with the remaining 5 µL ligation of 09/07.<br> |
| - | 0,8% gel : Cloning of cI failed. It seemed to have a problem with the biobrick’s size. May be the biobrick from the plate is wrong.<br> | + | 0,8% gel: Cloning of cI failed. It seemed to have a problem with the biobrick’s size. May be the biobrick from the plate is wrong.<br> |
<br> | <br> | ||
| - | Stand by because of a lack of plasmids backbones... | + | Stand by because of a lack of plasmids backbones...<br><br> |
</li> | </li> | ||
<li><span class="spantitle2">Week 7 (22-28 July)</span><br> | <li><span class="spantitle2">Week 7 (22-28 July)</span><br> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| - | < | + | </div> |
| + | </div> | ||
| + | |||
| + | <div class="accordeon"> | ||
| + | <h2 class="faq" title="Click to open the FAQ">August 2013</h2> | ||
| + | <div class="infos"> | ||
<ul class="circlearrow"> | <ul class="circlearrow"> | ||
<li><span class="spantitle2">Week 8 (29-4 August)</span><br> | <li><span class="spantitle2">Week 8 (29-4 August)</span><br> | ||
| - | We decide to use a new promoter (J23119) and also luxI gene (BBa-K081008) in order to concentrate our forces on the production and diffusion of AHL into gelose. | + | We decide to use a new promoter (J23119) and also luxI gene (BBa-K081008) in order to concentrate our forces on the production and diffusion of AHL into gelose.<br><br> |
</li> | </li> | ||
<li><span class="spantitle2">Week 9 (5-11 August)</span><br> | <li><span class="spantitle2">Week 9 (5-11 August)</span><br> | ||
| - | 5/08 : Transformation of J23119 and BBa-K081008 in DH5-1 cells.<br> | + | 5/08: Transformation of J23119 and BBa-K081008 in DH5-1 cells.<br> |
| - | 6/08 : Minipreps and assembly of J23119+K081008 in pSB1C3.<br> | + | 6/08: Minipreps and assembly of J23119+K081008 in pSB1C3.<br> |
7/08: Minipreps and verification of constructions by restriction. Restrictions with AvrII (in order to cut into J23119 biobrick) and PstI. We have one clone with the good restriction profile !<br> | 7/08: Minipreps and verification of constructions by restriction. Restrictions with AvrII (in order to cut into J23119 biobrick) and PstI. We have one clone with the good restriction profile !<br> | ||
| - | 8/08 : Overnight culture of Chromobacterium violaceum in 4 ml of LB with only 5 g/L of NaCl. Chromobacterium violaceum can detect AHL by producing violacein pigment.<br> | + | 8/08: Overnight culture of Chromobacterium violaceum in 4 ml of LB with only 5 g/L of NaCl. Chromobacterium violaceum can detect AHL by producing violacein pigment.<br> |
| - | 9/08 : Test of AHL production by J23119+K081008. | + | 9/08: Test of AHL production by J23119+K081008.<br><br> |
</li> | </li> | ||
<li><span class="spantitle2">Week 10 (12-18 August)</span><br> | <li><span class="spantitle2">Week 10 (12-18 August)</span><br> | ||
| - | Several experimentations to detect AHL production : <br> | + | Several experimentations to detect AHL production: <br> |
| - | - Incubation of Chromobacteirum + DH5-1 with J23119+BBA-K081008 on petri dishes during 24h at 35°C, 30°C and 25°C. | + | - Incubation of Chromobacteirum + DH5-1 with J23119+BBA-K081008 on petri dishes during 24h at 35°C, 30°C and 25°C. <br><br> |
</li> | </li> | ||
<li><span class="spantitle2">Week 11 (19-25 August) </span><br> | <li><span class="spantitle2">Week 11 (19-25 August) </span><br> | ||
Test of diffusion of AHL in LB gelose with commercial product (3-oxohexanoyl-homoserine lactone, sigma K3007). <br> | Test of diffusion of AHL in LB gelose with commercial product (3-oxohexanoyl-homoserine lactone, sigma K3007). <br> | ||
| - | Incubation 24 h at 25°C of Chromobacterium violaceum in presence of different amounts of AHL: 300 nmol, 30 nmol and 3 nmol. | + | Incubation 24 h at 25°C of Chromobacterium violaceum in presence of different amounts of AHL: 300 nmol, 30 nmol and 3 nmol.<br><br> |
</li> | </li> | ||
<li><span class="spantitle2">Week 12 (26-31 August) </span><br> | <li><span class="spantitle2">Week 12 (26-31 August) </span><br> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| + | </div> | ||
| + | </div> | ||
| - | < | + | <div class="accordeon"> |
| + | <h2 class="faq" title="Click to open the FAQ">September 2013</h2> | ||
| + | <div class="infos"> | ||
<ul class="circlearrow"> | <ul class="circlearrow"> | ||
| - | <li><span class="spantitle2"> | + | <li><span class="spantitle2">All month</span><br> |
| - | Modelling of diffusion of AHL into LB solid medium with the results of Chromobacterium experiences. | + | Modelling of diffusion of AHL into LB solid medium with the results of Chromobacterium experiences.<br><br> |
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| - | + | ||
| - | + | ||
| - | + | ||
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</li> | </li> | ||
</ul> | </ul> | ||
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</ul> | </ul> | ||
| + | </div> | ||
</div> | </div> | ||
<br> | <br> | ||
<br> | <br> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
| + | |||
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<p class="texte"> | <p class="texte"> | ||
<img src="https://static.igem.org/mediawiki/2013/3/3a/Back_arrow_-_14px.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/calendar/wet_lab">Back to Wet Lab</a></p> | <img src="https://static.igem.org/mediawiki/2013/3/3a/Back_arrow_-_14px.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/calendar/wet_lab">Back to Wet Lab</a></p> | ||
Latest revision as of 17:39, 4 October 2013
Calendar
Carry Characterization
June 2013
- Week 3 (24-30 June)
Amplification of new biobricks for cloning:
BBa_K081008: rbs Lux I,
BBa_I0462: rbs Lux R term,
BBa_C0061: cI,
BBa_R0065: pLux
BBa_K081014: rbs RFP term,
We tested a new method for midipreps using kits: alkaline lysis with spin columns. Results were really better in terms of samples purity. Cloning can start!!!
July 2013
- Week 4 (1-7 July)
The first necessary construction for carry characterization is the following:
Planning of cloning and study of the available methods for biobricks assembly.
Assembly of first biobricks thanks to the iGEM 3A Assembly method:
- strong prom/rbs + Lux I (698 bp) in PSB1K3
- pLux + rbs RFP term (936 bp) in PSB1A2
Restriction of parts, ligation and then transformation in DH5 α.
2 mL liquid cultures of 4 clones for each cloning.
Miniprep after 8 hours of incubation. The electrophoresis of the digested plasmids with EcoRI and PstI confirmed the presence of the wanted constructions for all clones (at 689 and 936 bp). A 1,5% gel was also run as reference to confirm the assembly. Small parts like promoters had been indeed added.
Cloning for carry characterisation keeps going. New constructions have been considered useful to study AHL diffusion.
New round of 3A cloning:
- strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3
- strong prom/rbs + rbs Lux R term (991 bp) in PSB1K3
- Week 5 (8-14 July)
2 mL liquid cultures of 6 clones for each cloning and then minipreps.
0,8% gel was run to confirm cloning success.
- correct insert for strong promoter strong rbs + Lux R terminator
- cloning failed for strong prom/rbs Lux I + rbs Lux R term (1634 bp) in PSB1C3
New try for cloning of 08/08.
Two new assembly with 3A method:
- strong promoter/rbs Lux R + pLux RFP in PSB1C3
- rbs cI term + pLux RFP in PSB1K3
2 mL liquid cultures of 6 clones for cI pLux RFP cloning and then minipreps.
Bacterial carpets were obtained for the two other clonings: transformation with the remaining 5 µL ligation of 09/07.
0,8% gel: Cloning of cI failed. It seemed to have a problem with the biobrick’s size. May be the biobrick from the plate is wrong.
Stand by because of a lack of plasmids backbones...
- Week 7 (22-28 July)
Verification by restriction of constructions. There are many problems with LuxI, LuxR and strong promoter.
August 2013
- Week 8 (29-4 August)
We decide to use a new promoter (J23119) and also luxI gene (BBa-K081008) in order to concentrate our forces on the production and diffusion of AHL into gelose.
- Week 9 (5-11 August)
5/08: Transformation of J23119 and BBa-K081008 in DH5-1 cells.
6/08: Minipreps and assembly of J23119+K081008 in pSB1C3.
7/08: Minipreps and verification of constructions by restriction. Restrictions with AvrII (in order to cut into J23119 biobrick) and PstI. We have one clone with the good restriction profile !
8/08: Overnight culture of Chromobacterium violaceum in 4 ml of LB with only 5 g/L of NaCl. Chromobacterium violaceum can detect AHL by producing violacein pigment.
9/08: Test of AHL production by J23119+K081008.
- Week 10 (12-18 August)
Several experimentations to detect AHL production:
- Incubation of Chromobacteirum + DH5-1 with J23119+BBA-K081008 on petri dishes during 24h at 35°C, 30°C and 25°C.
- Week 11 (19-25 August)
Test of diffusion of AHL in LB gelose with commercial product (3-oxohexanoyl-homoserine lactone, sigma K3007).
Incubation 24 h at 25°C of Chromobacterium violaceum in presence of different amounts of AHL: 300 nmol, 30 nmol and 3 nmol.
- Week 12 (26-31 August)
Modelling of diffusion of AHL into LB solid medium with the results of Chromobacterium experiences.
September 2013
- All month
Modelling of diffusion of AHL into LB solid medium with the results of Chromobacterium experiences.
