Team:INSA Toulouse/contenu/lab practice/results/polT7
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- | <h1 class="title1">Results - T7 Polymerase | + | <h1 class="title1">Results - T7 Polymerase characterization</h1> |
<h2 class="title2">Objective</h2> | <h2 class="title2">Objective</h2> | ||
- | Characterize the activity of T7 polymerase with an RFP reporter under a T7 promoter. Reminder: T7 polymerase is necessary in our system to activate the two T7 promoters in the AND1 and AND2 gate.< | + | <p class="texte">Characterize the activity of T7 polymerase with an RFP reporter under a T7 promoter. Reminder: T7 polymerase is necessary in our system to activate the two T7 promoters in the AND1 and AND2 gate.</p> |
<h2 class="title2">Conception</h2> | <h2 class="title2">Conception</h2> | ||
<p class="texte">The following constructions were designed:<br><br> | <p class="texte">The following constructions were designed:<br><br> | ||
<img src="https://static.igem.org/mediawiki/2013/0/07/Result_polT7_1.jpg" class="imgcenter" /><br><br> | <img src="https://static.igem.org/mediawiki/2013/0/07/Result_polT7_1.jpg" class="imgcenter" /><br><br> | ||
- | + | The coding sequence of T7 polymerase was extracted from the genome of <i>E. coli</i> BL21-DE3. Further cloning steps were necessary to add a promoter, a rbs and a terminator. The expected functioning is: | |
- | - Production | + | - Production of RFP if T7 polymerase is present (red colonies) |
- Absence of RFP if T7 polymerase is absent (white colonies)</p> | - Absence of RFP if T7 polymerase is absent (white colonies)</p> | ||
<h2 class="title2">Result</h2> | <h2 class="title2">Result</h2> | ||
- | <p class="texte"> | + | <p class="texte">The addition of a promoter and a rbs to the T7 polymerase gene was successful. However, we did not have time to go further and add the terminator to the construct.<br> Nevertheless, a new biobrick pT7-RFP was created!<br> |
- | Nevertheless, a new biobrick pT7-RFP was created! | + | |
- | Following is the electrophoresis gel showing that we obtained a fragment of expected length (829 bp) by restriction EcoRI/PstI:<br><br> | + | Following is the electrophoresis gel showing that we obtained a fragment of expected length (829 bp) by restriction with EcoRI/PstI:<br><br> |
<img src="https://static.igem.org/mediawiki/2013/f/f9/Result_polT7_2.jpg" class="imgcenter" /><br><br> | <img src="https://static.igem.org/mediawiki/2013/f/f9/Result_polT7_2.jpg" class="imgcenter" /><br><br> | ||
- | + | Verifications of the validity of the constructions were performed with visual inspection of the colours of the colonies. The pT7-RFP plasmid was transformed into competent BL21-DE3. Plates were supplemented or not with IPTG. White colonies were obtained on the Petri dishes with no IPTG and red colonies on the Petri dishes containing IPTG. </p> | |
- | White colonies | + | |
- | <img src="https://static.igem.org/mediawiki/2013/ | + | <img style="width:340px" src="https://static.igem.org/mediawiki/2013/9/94/Boite_rouge.jpg" class="imgcontentleft" /><br> |
- | <img src="https://static.igem.org/mediawiki/2013/ | + | <img style="width:340px" src="https://static.igem.org/mediawiki/2013/7/78/Boite_blanche.jpg" class="imgcontentright" /><div class="clear"></div> |
- | <h2 class="title2"> | + | <h2 class="title2">Discussion</h2> |
- | <p class="texte">The biobrick behave as expected. It was submitted to the registry | + | <p class="texte">The biobrick behave as expected. It was submitted to the registry as <a href=" http://parts.igem.org/Part:BBa_K1132045" target="_blank"> BBa_K1132045</a>.</p> |
Latest revision as of 15:13, 4 October 2013
Results - T7 Polymerase characterization
Objective
Characterize the activity of T7 polymerase with an RFP reporter under a T7 promoter. Reminder: T7 polymerase is necessary in our system to activate the two T7 promoters in the AND1 and AND2 gate.
Conception
The following constructions were designed:
The coding sequence of T7 polymerase was extracted from the genome of E. coli BL21-DE3. Further cloning steps were necessary to add a promoter, a rbs and a terminator. The expected functioning is:
- Production of RFP if T7 polymerase is present (red colonies)
- Absence of RFP if T7 polymerase is absent (white colonies)
Result
The addition of a promoter and a rbs to the T7 polymerase gene was successful. However, we did not have time to go further and add the terminator to the construct.
Nevertheless, a new biobrick pT7-RFP was created!
Following is the electrophoresis gel showing that we obtained a fragment of expected length (829 bp) by restriction with EcoRI/PstI:
Verifications of the validity of the constructions were performed with visual inspection of the colours of the colonies. The pT7-RFP plasmid was transformed into competent BL21-DE3. Plates were supplemented or not with IPTG. White colonies were obtained on the Petri dishes with no IPTG and red colonies on the Petri dishes containing IPTG.
![](https://static.igem.org/mediawiki/2013/9/94/Boite_rouge.jpg)
![](https://static.igem.org/mediawiki/2013/7/78/Boite_blanche.jpg)
Discussion
The biobrick behave as expected. It was submitted to the registry as BBa_K1132045.