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.