Difference between revisions of "Team:USP-Brazil/Circuit"

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<h1 style= "color:rgba(50, 0, 188, 0.8);">Circuit</h1>.
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<h1 style= "color:rgba(236, 27, 103, 0.8);">Assembling of the blue light sensor</h1>
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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.</br>
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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).</br>
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The resulting new plasmid (Part:BBa_K3095003) harbours all the components needed for the blue light sensor only.
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<img class="img-fluid" src="https://static.igem.org/mediawiki/2019/b/b2/T--USP-Brazil--BlueCircuit3.png">
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<figcaption>
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<b>Figure 3 </b>
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(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.
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<img class="img-fluid" src="https://static.igem.org/mediawiki/2019/6/64/T--USP-Brazil--BlueCircuit4.png">
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<b>Figure4></b>
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Complete plasmid with BBa_K3095003, also showing the primers used for confirmation.
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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
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<img class="img-fluid" src="https://static.igem.org/mediawiki/2019/1/1e/T--USP-Brazil--BlueCircuit5.png">
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<figcaption>
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<b>Figure>5</b>
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: (4)-strain with (BBa_K3095003) and pJFR4; (5)-strain with single blue light sensor (BBa_K3095003)  and pJFR5. Left plate: grown under LED light; Right plate: covered by aluminum foil. For the picture, plates were excited using a transilluminator (UV).
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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.</br>
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</br>
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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).
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</p>
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<figure>
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<img class="img-fluid" src="https://static.igem.org/mediawiki/2019/7/78/T--USP-Brazil--BlueCircuit6.png">
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<figcaption>
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<b>Figure>6</b>
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: (4)-strain with (BBa_K3095003) and pJFR4; (5)-strain with single blue light sensor (BBa_K3095003)  and pJFR5. Left plate: grown under LED light; Right plate: covered by aluminum foil. For the picture, plates were excited using a transilluminator (UV).
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</figcaption>
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</figure>
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<p style = "text-align: left">
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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.
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</p>
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Revision as of 00:57, 22 October 2019



Sticky Animated Navigation Bar

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.

Figure 3 (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.
Figure4> Complete plasmid with BBa_K3095003, also showing the primers used for confirmation.

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

Figure>5 : (4)-strain with (BBa_K3095003) and pJFR4; (5)-strain with single blue light sensor (BBa_K3095003) and pJFR5. 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 6).

Figure>6 : (4)-strain with (BBa_K3095003) and pJFR4; (5)-strain with single blue light sensor (BBa_K3095003) and pJFR5. Left plate: grown under LED light; Right plate: covered by aluminum foil. For the picture, plates were excited using a transilluminator (UV).

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.