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pLac-RBS-KillerRed
This BioBrick was obtained by PCR on an eukaryote vector containing the coding sequence of the KillerRed protein (generous gift of Stefan Dimitrov and Yohan Roulland, IAB Grenoble). A PCR allowed to flank it with the restriction sites KpnI and BamHI upstream and downstream. It was then ligated into the pQE30 vector(Qiagen), which contains a pLac promoter and RBS.
The KillerRed protein fits in our project in 2 ways: its fluorescence makes the transformed cells easily detectable and can be used to quantify the amount of live cells by our device. Its ROS-mediated killing is triggered by light, such that no chemical product has to be added in the medium to make it work and the process can be stopped as soon as the light is switched off.
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pLac-RBS-KillerRed
This BioBrick was obtained from BBa_K1141001 using PCR to amplify the part pLac-RBS-KillerRed. An overlapping PCR was performed to remove an EcoRI restriction site from the sequence in between pLac and RBS. The sequence was then flanked with the iGEM suffix and prefix.
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pLac-RBS-mCherry
This part contains the BioBricks BBa_R0010 and BBa_J06702.
It was built with standard assembly.
In the project this BioBrick was used as a negative control of KillerRed as mCherry has similar excitation and emission peaks without being cytotoxic (very low ROS production). This BioBrick also allowed us to characterize the two BioBricks BBa_R0010 and BBa_J06702 with new data.
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Green sensor-GFP
Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab.
This part was used in the light controlled module it has been built to characterize the green sensor.
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Red sensor-RFP
Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab.
This part is a negative control that does not produce as much ROS a KillerRed.
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Red Sensor-KillerRed
Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab.
This BioBrick takes part in the light controlled KillerRed concentration module. It is the enhanced version of BBa_K1141001 with a light sensitive promoter.
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pBad-RBS-sspB
This part contains the BioBricks pBAD K206000 and sspB K174000, the RBS has been added via PCR on sspB, the rest of the BioBrick was built by standard assembly.
This BioBrick is a part of the degradation module , it enhances the degradation rate of any protein tagged with an ''ssrA''-tag.
