Team:INSA Toulouse/contenu/lab practice/results/polT7
From 2013.igem.org
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:![](https://static.igem.org/mediawiki/2013/0/07/Result_polT7_1.jpg)
Coding sequence of T7 polymerase was extracted from the genome of E.Coli BL21-DE3. Cloning are 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
Cloning to add a promoter and a rbs to T7 polymerase was a success but Cloning for adding a terminator to T7 polymerase was never achieved. 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:![](https://static.igem.org/mediawiki/2013/f/f9/Result_polT7_2.jpg)
Further verifications were done with phenotypic test. Plasmid was transformed into competent BL21-DE3. BL21-DE3 is the strain containing T7 polymerase under of Lac promoter (inductible with IPTG). White colonies was obtained into petri dish and red colonies into petri dish with IPTG
![](https://static.igem.org/mediawiki/2013/4/43/Result_polT7_3.jpg)
![](https://static.igem.org/mediawiki/2013/3/3e/Result_polT7_4.jpg)
Dicussion
The biobrick behave as expected. It was submitted to the registry (lien BBa_K1132045). Further characterization can be done concerning the activity of T7 polymerase, looking to the production of RFP during time.