Difference between revisions of "Team:Bielefeld-CeBiTec/Results/toolbox/photolysis"

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<h2> Change of structure of 2-nitrophenylalanine due to UV-light</h2>
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To test if the structure of 2-NPA can be changed by irradiation with our LED panel, an <i>in vitro</i> test was conducted. LB media supplemented with 1 mM 2-NPA was used for absorption measurements over 4 hours while irradiation with UV-light in a 96-well microtiter plate. A high absorption in the UV spectrum (< 300 nm) was observed, which makes sense since 2-NPA absorbs the UV-light to do the self-cyclization reaction (Figure 8). Over time a constant increase in the absorption spectrum at approximately 340 nm could be noticed, which indicates the emergence of a chemical component due to the irradiation process. We are confident that this compound is the 2-NPA in its cinnoline form, as proposed by <i>Peters et al.</i>, which leads to a shift in the absorption spectrum.
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<p class="figure subtitle"><b>Figure 8:</b>Changes in the absorption spectrum of 2-NPA in LB media while irradiated at 367 nm for 240 minutes. The emerging peak at ~ 340 nm indicates the change in the structure of 2-NPA from its native form, to the self-cyclized form.<p>
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Therefore, we were able to confirm that the LED panel is suitable for the induction of the photolysis of 2-NPA. Furthermore, to the best of our knowledge, we conducted the most detailed documentation of the changes in the absorption spectrum of 2-NPA during the self-cyclization process to its cinnoline form.
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<h2>Cleavage of the fusion protein</h2>
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To prove that photolysis of 2-NPA is also possible when incorporated into a protein, three transformants of <i>E.coli</i> BL21(DE3) were cultivated. The first one contains the part BBa_K2201320 which expresses the whole fusion protein of GFP and streptavidin. The second one contains the part BBa_K2201321 and produces the GFP-unit of the fusion protein due to the amber stop codon in the linker. The third one is a cotransformant of BBa_K2201220 (2-NPA-RS) and BBa_K2201321 and therefore should produce the whole fusion protein with 2-NPA in the linker. The cells were cultivated in 50 mL LB-media (for the cotransformants supplemented with 1 mM 2-NPA) in 500 mL shaking flask for 16 hours at 37 °C and 150 rpm. The cells were harvested and lysed after the cell lyses protocol for SDS-Page. Two Samples of the cell lysate of the cotransformants were transferred to a 96-well microtiter plate and irradiated with the LED panel at 367 nm and 100 % brightness . After irradiation the samples were transferred to a SDS-Page (Figure 9).
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<p class="figure subtitle"><b>Figure 9:</b>SDS-Page of the whole fusion protein as positive control 1, GFP-unit as positive control 2, and two samples of the irradiated fusion protein containing 2-NPA after 1 hour and 5 hours of irradiation with UV-light. In dark green are the bands of the whole fusion protein. In purple the bands of the 2-NPA-RS In light green the GFP-unit of the fusion protein and in yellow the bands of the cleaved streptavidin-Tag.<p>
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Figure 9 shows the bands of the whole fusion protein at approximately 45 kDa for the positive control 1 and the two samples. Furthermore, the bands of the GFP-units can be seen at approximately 25 kDa and the bands of the 2-NPA-RS at ~ 35 kDa. At the bottom of the SDS-page the bands of the cleaved streptavidin-tags of the two samples can be observed. Unfortunately, there was an uneven running front which led to a shift of the samples compared to the marker. Therefore, a western blot was performed (Figure 10) with specific anti-GFP and anti-strep antibodies to verify that the bands seen in Figure 9 are truly the peptides we expected.
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<img class="figure image" src="https://static.igem.org/mediawiki/parts/f/ff/T--Bielefeld-CeBiTec--YKE_Westernblot_photolysis1.png">
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<p class="figure subtitle"><b>Figure 10:</b>Western Blot of a SDS-Page similar to Figure 9 marked with anti-GFP antibodies to determine the bands marked in Figure 9. It proves that the low bands in Figure 9 are indeed cleaved streptavidin-Tags.<p>
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The western blot confirmed our expectations. The bands of the whole fusion protein, of the GFP-units and the cleaved streptavidin-tags are clear to see. The GFP-bands are very thick compared to the others, which cannot be explained by the photolysis itself. It seems like the 2-NPA-RS is not very effective in loading 2-NPA to the amber tRNA, which leads to a high amount of incomplete expression of the fusion protein. The low efficiency of our 2-NPA-RS was confirmed by our synthetase-test system (K2201343). Therefore, we started modeling and selection of a new aaRS, able to incorporate 2-NPA to provide a better synthetase to the iGEM community.
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Revision as of 16:47, 26 October 2017

Photolysis

Design of 2-NPA-RS

At first, we designed a gene synthesis at IDT based on the selection experiment from Peters et al. (2009) to get an aminoacyl-tRNA-synthetase to incorpate 2-nitrophenylalanine. An alignment of the protein sequences of the native M. jannaschii TyrRS which was the basis of the selection experiment and the used 2-Nitrophenylalanine-synthetase is shown in Figure 1. They differ in ten amino acids.

Figure 1: Alignment of the protein sequences of the M. jannaschii tyrosyl synthetase and the 2-Nitrophenylalanine synthetase designed by Peters et al.

Cloning of this NPA-RS in pSB1C3 and pSB3T5

We then used the protein sequence of the clone with the highest fidelity for 2-NPA and translated it into a gene sequence which was then codon optimized for E.coli. We designed it with matching overhangs of 35bp to a linearized ONBY-Part (K1416000) in pSB1C3 to get the sequence in a matching expression cassette. The psb1c3 backbone is a high copy plasmid and for an adequate usage of the aaRS it is needed on a low copy plasmid. We so used BioBrick assembly to get the insert of the new 2-NPA-Part (K2201200) in the low copy plasmid of pSB1K3 (Figure 2) for further use. The Insert of K2201200 in the low copy plasmid is available on request at the CeBiTec.

Figure 2: Two Plasmids we created for our toolkit for the iGEM community. Left: 2-NPA-RS in the pSB1C3 high copy plasmid (K2201200). Right: 2-NPA-RS in the pSB3T5 low copy plasmid (available on request) at the CeBiTec.

Design of fusion protein

We also designed two fusion proteins to verify the incorporation and functionality of the 2-NPA (Figure 3). Plasmid I (K2201320) codes for a simple GFP-streptavidin fusion protein connected by a gly-gly-ser-linker. Plasmid II (K2201321) is homologous to plasmid I but has an amber codon in the middle of the linker. If transformed in E.coli BL21(DE3) (1) only the GFP-unit will be expressed (B). If cotransfromed with an aaRS for a noncanonical amino acid but without feeding the specific ncAA (2) the aaRS will incorporate other amino acids profoundly phenylalanine in the linker (C). If cotransformed and with the 2-NPA in the culture media (3) the fusion protein will be expressed with 2-NPA in the linker (D). The fusion protein can then be irradiated by light of a wavelength of 365nm (4) to induce the cleavage of the fusion protein to its GFP-unit (E) and the streptavidin-unit (F).

Figure 3: Design of two plasmids for fusion proteins. I) Plasmid (K2201320) for reference protein of GFP (green) a linker (purple) and streptavidin (yellow) (A). II) Plasmid (K2201321) for the application protein with Amber-codon (black star) in the linker for three different protein variants after expression. 1: Solely expression leads to GFP-unit and linker to the Amber-codon (B). 2: Cotransformed with a 2-NPA-RS (K2201200) without 2-NPA leads to a fusion protein with an unspecific amino acid (presumably phenylalanine, red star) in the linker (C). 3: Cotransformed with 2-NPA-RS and 2-NPA leads to the functional fusion protein with 2-NPA (purple star) in the linker (D). 4: Irradiation of protein D leads to a cleavage of the fusion protein in the GFP-unit (E) and the streptavidin unit (F).

Proof of incorporation of AS at Amber-codon when cotransformed with NPA-RS

To proof the expression of the 2-NPA-RS in K2201200 we transformed this part, the ONBY-Part (K1416000) and a plasmid only containing the coding sequence of the fusion protein in E.coli BL21(DE3). The three transformants were cultivated in LB-Media for 16 hours at 37°C and 150 rpm. The cultures were harvested and the pellets lysed after the standard protocol of cell lysis in lysis buffer. Afterwards 15 µl of the samples were then transferred to an SDS-Page (Figure 4). There were two big bands present at approximately 35 kDA at the 2-NPA and the ONBY sample which correlates with the expected masses of the synthetases. We so are sure that the 2-NPA-RS in part K2201200 is well expressed when transformed in a matching E.coli strain like BL21(DE3).

Figure 4: SDS-Page of the expressed 2-NPA-RS (left) from K2201200 with ONBY-RS from K1416000 as positive control (middle) and the basic protein expression of BL21(DE3) as negative control (right).

To proof that our 2-NPA-RS is able to incorporate amino acids to the amber codon the parts was cotransformed with plasmid II (K2201321) in E.coli BL21(DE3) along with plasmid I (K2201320) and plasmid II was also transformed separately. After cultivation and cell lysis as mentioned above (in which one culture of the cotransformants was cultivated with 1mM and another without 2-NPA) the samples were transferred on an SDS-Page and a western-blot with anti-GFP-antibodies was performed (Figure 5).

Figure 5: Western blot with GFP-antibodies of the four different fusion proteins variants (figure 3) as proof of the functionality of the 2-NPA-RS. The band marked with a * is weak because of degradation of the fusion protein while the storage. The bands at approximately 45,0 kDa mark the mass of the whole fusion protein (~ 40,9 kDa), the bands at approximately 25,0 kDa mark the GFP-unit (~ 27,0 kDa) of the fusion protein.

Figure 5 shows that only the whole fusion protein of plasmid II was expressed (A) and only the GFP-unit of plasmid II if not cotransformed with a matching aaRS (B). The fusion protein of plasmid II was fully expressed when cotransformed with the 2-NPA-RS and when cultivated with (D) and without (C) 2-NPA in the media.

Permeability of Microwellplate by irradiation of 365nm

To proof which micro well plates are suitable for the irradiation with our LED-Panel we made a little absorption study of three plates that were available. A black and a transparent 96-well plate from nunc and a transparent well plate from greiner (Figure 6).

Figure 6: Three microwell plates tested for their suitability for the irradiation with the LED-panel. Left: Black nunc plate. Middle: Transparent nunc plate. Right: Transparent greiner plate.

The absorption of the empty plates were measured in a the Tecan Infinite M200 (Roche) at wavelengths from 300 to 450 nm (Figure 7, left). The measured adsorption values were converted to the amount of light that passes the plates in percentage (Figure 7, right). Figure 7 shows that all plates are suited for the application with the LED-panel. At 365 nm 80 % of the light permits both of the transparent plates and 87 % of the light permits the black nunc plate. A small fraction of light will always be absorbed but the amount of light that permits the plates should be high enough for the photlysis and the photoswitchung experiments.

Figure 7: Results of the irradiation test of the three microwell plates: Left: Extinction of the plates for light of the wavelengths from 300 to 450 nm. Right: Calculated light-permeability in % of the three tested plates. At 365 nm wavelength 87% of the light permits the black nunc plate, 80 % permits the transparent nunc plate and 76 % permits the transparent greiner plate.

Change of structure of 2-nitrophenylalanine due to UV-light

To test if the structure of 2-NPA can be changed by irradiation with our LED panel, an in vitro test was conducted. LB media supplemented with 1 mM 2-NPA was used for absorption measurements over 4 hours while irradiation with UV-light in a 96-well microtiter plate. A high absorption in the UV spectrum (< 300 nm) was observed, which makes sense since 2-NPA absorbs the UV-light to do the self-cyclization reaction (Figure 8). Over time a constant increase in the absorption spectrum at approximately 340 nm could be noticed, which indicates the emergence of a chemical component due to the irradiation process. We are confident that this compound is the 2-NPA in its cinnoline form, as proposed by Peters et al., which leads to a shift in the absorption spectrum.

Figure 8:Changes in the absorption spectrum of 2-NPA in LB media while irradiated at 367 nm for 240 minutes. The emerging peak at ~ 340 nm indicates the change in the structure of 2-NPA from its native form, to the self-cyclized form.

Therefore, we were able to confirm that the LED panel is suitable for the induction of the photolysis of 2-NPA. Furthermore, to the best of our knowledge, we conducted the most detailed documentation of the changes in the absorption spectrum of 2-NPA during the self-cyclization process to its cinnoline form.

Cleavage of the fusion protein

To prove that photolysis of 2-NPA is also possible when incorporated into a protein, three transformants of E.coli BL21(DE3) were cultivated. The first one contains the part BBa_K2201320 which expresses the whole fusion protein of GFP and streptavidin. The second one contains the part BBa_K2201321 and produces the GFP-unit of the fusion protein due to the amber stop codon in the linker. The third one is a cotransformant of BBa_K2201220 (2-NPA-RS) and BBa_K2201321 and therefore should produce the whole fusion protein with 2-NPA in the linker. The cells were cultivated in 50 mL LB-media (for the cotransformants supplemented with 1 mM 2-NPA) in 500 mL shaking flask for 16 hours at 37 °C and 150 rpm. The cells were harvested and lysed after the cell lyses protocol for SDS-Page. Two Samples of the cell lysate of the cotransformants were transferred to a 96-well microtiter plate and irradiated with the LED panel at 367 nm and 100 % brightness . After irradiation the samples were transferred to a SDS-Page (Figure 9).

Figure 9:SDS-Page of the whole fusion protein as positive control 1, GFP-unit as positive control 2, and two samples of the irradiated fusion protein containing 2-NPA after 1 hour and 5 hours of irradiation with UV-light. In dark green are the bands of the whole fusion protein. In purple the bands of the 2-NPA-RS In light green the GFP-unit of the fusion protein and in yellow the bands of the cleaved streptavidin-Tag.

Figure 9 shows the bands of the whole fusion protein at approximately 45 kDa for the positive control 1 and the two samples. Furthermore, the bands of the GFP-units can be seen at approximately 25 kDa and the bands of the 2-NPA-RS at ~ 35 kDa. At the bottom of the SDS-page the bands of the cleaved streptavidin-tags of the two samples can be observed. Unfortunately, there was an uneven running front which led to a shift of the samples compared to the marker. Therefore, a western blot was performed (Figure 10) with specific anti-GFP and anti-strep antibodies to verify that the bands seen in Figure 9 are truly the peptides we expected.

Figure 10:Western Blot of a SDS-Page similar to Figure 9 marked with anti-GFP antibodies to determine the bands marked in Figure 9. It proves that the low bands in Figure 9 are indeed cleaved streptavidin-Tags.

The western blot confirmed our expectations. The bands of the whole fusion protein, of the GFP-units and the cleaved streptavidin-tags are clear to see. The GFP-bands are very thick compared to the others, which cannot be explained by the photolysis itself. It seems like the 2-NPA-RS is not very effective in loading 2-NPA to the amber tRNA, which leads to a high amount of incomplete expression of the fusion protein. The low efficiency of our 2-NPA-RS was confirmed by our synthetase-test system (K2201343). Therefore, we started modeling and selection of a new aaRS, able to incorporate 2-NPA to provide a better synthetase to the iGEM community.