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.
Approach A
Our main project, the Approach A, was fully dependent on the synthesis of all of the components, which summed over 8.2 kb of DNA. We divided them into 5 blocks; 3 were ordered from IDT and 2 from Twist Biosciences. Golden Gate (GG) assembling was the chosen strategy to ligate the blocks into pGGA vector.
In order to make a safety stock of the fragments, each block was cloned into pCR-Blunt (Thermo). At this point, we failed to clone one of the fragments in the vector, even though we have tried several times.
On top of that, we also tried to make a GG reaction using the linear DNA that we’ve received from the synthesis. We have screened over 1500 colonies using the standard protocol, and even after making small variations, we could not find the right construct. The main hypothesis for this problem was that we were not able to clone one of the blocks. We believe that the fragment was already degraded when we received it. We contacted IDT and they were kind enough to resend us the fragment, free of charges. Unfortunately, this block has not arrived yet; therefore, we could not proceed with this approach.
Approach B
For the construction of Approach B circuit (BBa_K3095011), all of the five blocks were ordered from Twist Bioscience. Since we wanted to gather all of the fragments into a single vector (pGGA), we chose to do it by Golden Gate assembly. Therefore, the fragments were designed to have BsaI restriction sites at the 5’ end, and each of them had base complementarity with neighboring fragments. The Golden Gate reaction was used to transform DH10B competent cells.
Owing to the fact that there is a constitutive promoter expressing a fluorescent protein (mTagBFP2) in Approach B circuit, we took opportunity of this feature to screen all of the transformed colonies using black light. Blue fluorescent cells are expected to have at least two of the fragments inserted in pGGA, but the complete construction is also possible. We found only one colony that had blue fluorescence, and it was picked to be confirmed by digestion (XhoI). Later, PCR was done to confirm whether the order of insertion was correct (Figure 7), primers PB1 and T7, should result in amplification of about 500 bp; PB2 and PB3 (about 500 bp); PB4 and PB2 (about 1400 bp); PB6 and PB5 (about 500 bp); SP6 and PB7 (about 1400 bp).
Once the results showed everything was right, we tried to subclone the whole Approach B circuit into pJN105 vector. Because of lack of time, we did not manage to finish this construct, but we are very close to do so.
However, we did a few experiments to validate and characterize the new part mTagBFP2(BBa_K3095008). Fluorescence microscopy was performed to detect bacteria expressing the blue fluorescent protein (Figure 9), and we also quantified the level of fluorescence by a spectrofluorometer (Figure 10). The excitation wavelength at 399 nm, and emission at 454 nm were set according to the data found in FPbase.
Although mTagBFP2 was under regulation of a constitutive promoter (pJ23100), the fluorescence was not very strong, and sometimes even hard to spot with the naked eye. However, it was very evident when using a spectrofluorometer or fluorescence microscope. This feature, summed to the low wavelength of emission/excitation, makes it very usefull to use as a constitutive reporter, in order to normalize the expression of other reporters, since the intensity and its fluorescence is not likely to interfere with the measurement of other common reporters.
Although mTagBFP2 was under regulation of a constitutive promoter (pJ23100), the fluorescence was not very strong, and sometimes even hard to spot with the naked eye. However, it was very evident when using a spectrofluorometer or fluorescence microscope. This feature, summed to the low wavelength of emission/excitation, makes it very usefull to use as a constitutive reporter, in order to normalize the expression of other reporters, since the intensity and its fluorescence is not likely to interfere with the measurement of other common reporters.