Part:BBa_K3095011:Experience

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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 1), 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).

Figure1 A) – digestion of BBa_K3095011 cloned in pGGA vector. XhoI has two sites for this plasmid. when the digestion occurs BBa_K3095011 (about 4600 bp) is released from the backbone (2100 bp). (B) – PCR to check the right orientation of each block. The amplification at the sizes shown in the gel confirms the expected construction.

Figure 2 Representation of BBa_K3095011 cloned in pGGA showing the primers used for confirmation.

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 3), and we also quantified the level of fluorescence by a spectrofluorometer (Figure 4). The excitation wavelength at 399 nm, and emission at 454 nm were set according to the data found in FPbase.

Figure 3 Comparison between DH10B strain transformed with pGGA + BBa_K3095011 (A) or just pGGA (B) under fluorescence microscope.

Figure 4 Representation of BBa_K3095011 cloned in pGGA showing the primers used for confirmation.

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