Circuit
.Assembling of the blue light sensor
Because our Project only needed one input, we decided to use the blue light sensor from the Jesus & Voigt et al 2017 construct. However, since the genes for the complete circuit of the blue light are spread along pJFR1 and pJFR2, we chose to merge the genes into a single vector for the sake of simplicity. Firstly, we used pJFR2 as backbone and excised the unnecessary gene from the green light sensor (cgg). Later, the fragments coding for T7 polymerase (core) and YF1-fixJ were HF-PCR amplified and inserted by restriction/ligation cloning. Colony PCR was performed to screen and detect the right clones. To confirm the first insertion, primers ‘conf1 fw’ and ‘conf1 rv’ were used, and ‘teste fw’ and ‘conf1 rv’ for the second one (Figure 3). The resulting new plasmid (Part:BBa_K3095003) harbours all the components needed for the blue light sensor only.
To test whether the new construct was working properly, we transformed E. coli DH10B strain with (BBa_K3095003) and pJFR4, which carries the output for the blue light sensor, a blue fluorescent protein (BFP). The strain carrying (BBa_K3095003) and pJFR4 was streaked in plates and grown over the LEDbox, which emits light at 450 nm. The Figure 5, bellow shows that only the strain transformed with (BBa_K3095003) and pJFR4 emits blue fluorescence when cultivated under blue LED
To further test the blue light sensor, and inspired by Jesus & Voigt et al 2017 article, we decided to print images into LB plates using this system. To do so, we have built a fully functional projector made of card box, and we took this opportunity to advertise our best partners: BioLambda and IPT. For this task, we used a strain containing (BBa_K3095003) and pJFR5. pJFR5 has a promoter inducible by blue light, which activates the transcription of bFMO. The resulting protein converts indole to indigo (dark blue pigment) and its staining is visible on the plate (Figure 6).
By showing the results in the Figure 5 and 6, we demonstrate that our new construct (BBa_K3095003), which carries all the necessary genes for blue light sensing, is working properly.