Characterisation of BBa_J04450

iGEM Grenoble 2017 team worked on BBa_J04450.
This part has been designed by Tamar Odle in 2005. This is an RFP coding device.
Figure 1 : Scheme of the BBa_J04450 part, from iGEM parts database
The colonies of bacteria transformed with this plasmid are clearly red under natural light after about 18 hours. Smaller colonies are visibly red under UV. The RFP part does not contain a degradation tag and the RBS is strong. This part is commonly used, but can fail if the system contains LacI or CAP protein. (Informations from iGEM website, “parts” section).
The promoter of mRFP1 gene in each plasmid is supposed to be inducible (with IPTG) but a strong leak of RFP production could be observed. (fig 2)
Figure 2 : Observation of the leak of the promoter. A : Bacteria transformed or not with BBa_J04450 sequence, naked eye, white light. B. Bacteria without mRFP1 gene, excited at 546nm. C. Bacteria with mRFP1 gene, excited at 546nm. Red fluorescence is notable. Photos taken thanks to SnapLab, the biodetection kit of iGEM Grenoble 2017 (fig 3).
Figure 3 : SnapLab, the detection kit made by Grenoble-Alpes iGEM Team.

Does IPTG induction bring forward and/or increase RFP production ?

A difference of fluorescence was evaluated, either on the intensity either on the time of apparition of the fluorescence. The following experiment has been designed in this aim.


Results showed IPTG had an importance in RFP production. Indeed, we can see that IPTG allows a shorter rise of fluorescence. It could be interesting to repeat this experience with different IPTG concentrations, to see whether the delay of apparition of the red fluorescence would decrease with a higher amount of inducer.

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