Part:BBa_K3095003:Experience

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USP-Brazil 2019 Characterization

Methods

1. Parts removal and plasmid reconstruction.

Utilizing 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 fisrt insertion, primers ‘conf1 fw’ and ‘conf1 rv’ were used, and ‘teste fw’ and ‘conf1 rv’ for the second one (Figure 1). The created construct can be visualised in the figure 2.

Figure 1: (A) – Gel purification from pJFR2(∆cgg), the size was about 4300 bp. (B) – ColonyPCR to detect the insertion of T7 polymerase (core)’s gene. The right cloning should give a fragment of about 2600 bp, which is shown in the lanes 2 and 4. (C) – ColonyPCR to detect the insertion of YF1-fixJ fragment. The successful construct should be around 2500 bp, which is shown in the lanes 7 and 10.

Figure 2: Complete plasmid with BBa_K3095003, also showing the primers used for confirmation.

Results

For test the our construction, we utilised the light-regulated promoter pT3 (BBa_K2598026) with a BFP reporter. The strain carrying (BBa_K3095003) and pJFR4 was streaked in plates and grown over the LEDbox, which emits light at 450 nm. The Figure 3, bellow shows that only the strain transformed with (BBa_K3095003) and pT3-BFP emits blue fluorescence when cultivated under blue LED.

Figure 3: (4)-strain with (BBa_K3095003) and pT3-BFP; (5)-strain with single blue light sensor (BBa_K3095003) and pJFR5 - pT3 without BFP. Left plate: grown under LED light; Right plate: covered by aluminum foil. For the picture, plates were excited using a transilluminator (UV).

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 4).

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