Difference between revisions of "Part:BBa K5398600:Experience"
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We use TyrVs to oxidize tyrosine residues in TRn4-mfp5(BBa_K5398020) to L-DOPA,thereby endowing it with adhesion ability.
 
We use TyrVs to oxidize tyrosine residues in TRn4-mfp5(BBa_K5398020) to L-DOPA,thereby endowing it with adhesion ability.
 
===Characterization===
 
===Characterization===
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====Plasmid Construction====
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To validate the functionality of the tyrosinase TyrVs, we designed bacteria expressing TyrVs.We constructed the pET-SUMO-TyrVs vector, after culturing at 16°C for 20 h, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis.  
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We considered cloning TyrVs into the pET-PC-SUMO vector to explore the potential for enhancing its expression level. We constructed the pET-PC-SUMO-TyrVs vector and transformed it into <i>E. coli</i>BL21(DE3).
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         <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/sumo-tyrvs.webp" width="400" height="auto" alt="Protein purification">
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         <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/sumo-tyrvs-mizuo-new.webp" width="400" height="auto" alt="Protein purification">
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        <p><b>Fig. 2 | Plasmid pET-PC-SUMO-TyrVs construction results.</b></p>
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         <p><b>Fig. 2 | Expression of recombinant TyrVs in <i>E. coli</i>BL21(DE3) with pET-PC-SUMO-TyrVs.</b></p>
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<p>a.Expression plasmids of TyrVs. b.PCR results of pET-PC-SUMO-TyrVs. Line 1: Marker. Lines 2-3:Vector;Lines 4-5:Gene.</p>
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<p>Lane 1: Marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells respectively.</p>
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====Protein expression====
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a single colony
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from a freshly streaked plate of the cells was cultured in 5 mL of LB
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medium with 25 μg/mL Ampicillin at 37℃ overnight. The secondary
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cultures were prepared with 1% inoculum in 50 mL of LB medium with 25 μg/mL Ampicillin. Cultures were
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then incubated at 37℃ and 200 rpm until the optical density at 600 nm
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(OD<sub>600</sub>) reached 0.6–0.8. 1 mM IPTG was added to induce production of recombinant proteins and
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cultures were further cultivated at 16℃ and 200 rpm for 20 h. The cells
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were collected by centrifugation at 6000 ×g at 4℃ for 20 min.The recombinant cells were harvested by centrifugation and re-suspension in lysis buffer(10 mM imidazole, 50 mM Tris-HCl, 500 mM NaCl, pH 8.0)and lysed on ice by sonication.Sonicated samples were centrifuged at 12,000 ×g at 4 ◦C for 20 min to obtain insoluble and soluble fractions.After protein extraction, different proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue.
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         <p><b>Fig. 3 | Expression of recombinant TyrVs in <i>E. coli</i>BL21 (DE3) with pET-PC-SUMO-TyrVs.</b></p>
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<p>Lane 1: Marker. lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively;lanes 5 to 7: whole-cell lysate, supernatant and pellet from induced cells respectively.</p>
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====Western blotting====
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Western bolotting revealed that after induction with IPTG, TyrVs was primarily expressed in its soluble form.
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        <p><b>Fig. 4 | Western blotting analysis recombinant TyrVs in <i>E. coli</i>BL21 (DE3) with pET-PC-SUMO-TyrVs.</b></p>
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<p> Lane 1-3:whole-cell lysate,pellet and supernatant from induced cells with 0.5 mM IPTG respectively.</p>
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         <p><b>Fig. 5 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.</b></p>
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         <p><b>Fig. 3 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.</b></p>
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<p>Lane 1: Marker. Lane 2: Lysis Buffer. Lane 3: Supernatant. Lane 4: 20 mM Imidazole. Lane 5: 50 mM Imidazole. Lane 6: 150 mM Imidazole. </p>
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<p>Lane 1: Marker; Lane 2: Lysis Buffer; Lane 3: Supernatant; Lane 4: 20 mM Imidazole; Lane 5: 50 mM Imidazole; Lane 6: 150 mM Imidazole. </p>
 
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====Enzyme activity test====
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We conducted tests on the reactions from tyrosine to dopaquinone and from L-DOPA to dopaquinone. The experiment of enzymatic reaction from tyrosine to dopaquinone was conducted at 37℃ with an enzyme concentration of 0.1 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (V<sub>max</sub>) were 456.8 μmol/L and 0.31 μmol/L·s, respectively. The experiment of enzymatic reaction from L-DOPA to dopaquinone was conducted at 37°C with an enzyme concentration of 0.2 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (V<sub>max</sub>) were 8787 μmol/L and 0.86 μmol/L·s, respectively.
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We dialyzed the extracted SUMO-TyrVs for 24 hours, followed by diluting it 10,000-fold for enzymatic activity assays. In a 96 Well Cell Culture Plates, we prepared different concentrations of tyrosine and L-DOPA solution, added the diluted SUMO-TyrVs, and measured the change in OD475 over the first 5 minutes using a microplate reader.
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        <p><b>Fig. 6 | The 96 Well Cell Culture Plates of tyrosinase TyrVs.</b></p>
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<p>a.The experiment of enzymatic reaction from tyrosine to dopaquinone. b.The experiment of enzymatic reaction from L-DOPA to dopaquinone. </p>
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The data were processed to generate a Michaelis-Menten curve and a Lineweaver-Burk plot. The experiment of enzymatic reaction from tyrosine to dopaquinone was conducted at 37°C with an enzyme concentration of 0.1 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 456.8 μmol/L and 0.31 μmol·L<sup>-1</sup>·s<sup>-1</sup>, respectively. The experiment of enzymatic reaction from L-DOPA to dopaquinone was conducted at 37°C with an enzyme concentration of 0.2 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 8787 μmol/L and 0.86 μmol·L<sup>-1</sup>·s<sup>-1</sup>, respectively.
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         <p><b>Fig. 7 | The activity assay results of tyrosinase TyrVs</b></p>
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         <p><b>Fig. 4 | The activity assay results of tyrosinase TyrVs</b></p>
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<p>a-b.Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. c-d.Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments. </p>
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<p><b>a-b.</b> Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. <b>c-d.</b> Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments. </p>
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====Mathematical modeling Analysis====
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Tyrosinase exhibits dual catalytic properties, capable of catalyzing the conversion of tyrosine to L-DOPA and L-DOPA to dopaquinone. We analyzed through mathematical modeling to determine how to maximize the oxidation of tyrosine to L-DOPA. We selected multiple sets of parameters for fitting, and the goodness of fit $R ^ 2 $was 0.9962, indicating a good fitting effect. We incorporated appropriate fitting parameters into the established model and determined that the optimal reaction time is approximately 130 seconds, at which point the production of L-DOPA reaches its peak. To demonstrate that the reaction can proceed stably under conventional conditions, we introduced perturbations in each reaction channel.Under different disturbance conditions, the trend of dopamine quantity changes is similar, and the yield fluctuation is small, our reaction system has strong environmental adaptability and stability.
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        <p><b>Fig. 7 | The activity assay results of tyrosinase TyrVs</b></p>
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<p>a.Data fitting results. b.Changes in the concentrations of various substances in the reaction system. c.Changes in the concentration of substances in the system after adding disturbances.</p>
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<br><br><br><br>
 
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Revision as of 11:35, 1 October 2024

Applications of BBa_K5398610

We use TyrVs to oxidize tyrosine residues in TRn4-mfp5(BBa_K5398020) to L-DOPA,thereby endowing it with adhesion ability.

Characterization

To validate the functionality of the tyrosinase TyrVs, we designed bacteria expressing TyrVs.We constructed the pET-SUMO-TyrVs vector, after culturing at 16°C for 20 h, extracted the proteins for SDS-PAGE and Coomassie Brilliant Blue staining analysis.

Protein purification

Fig. 2 | Expression of recombinant TyrVs in E. coliBL21(DE3) with pET-PC-SUMO-TyrVs.

Lane 1: Marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells respectively.

We purified SUMO-TyrVs using a HiTrap Ni-NTA column. The purified protein was verified by SDS-PAGE and was found to be present in the 50 mM imidazole elution fraction.
Protein purification

Fig. 3 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.

Lane 1: Marker; Lane 2: Lysis Buffer; Lane 3: Supernatant; Lane 4: 20 mM Imidazole; Lane 5: 50 mM Imidazole; Lane 6: 150 mM Imidazole.

We conducted tests on the reactions from tyrosine to dopaquinone and from L-DOPA to dopaquinone. The experiment of enzymatic reaction from tyrosine to dopaquinone was conducted at 37℃ with an enzyme concentration of 0.1 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 456.8 μmol/L and 0.31 μmol/L·s, respectively. The experiment of enzymatic reaction from L-DOPA to dopaquinone was conducted at 37°C with an enzyme concentration of 0.2 μg/mL. The calculated Michaelis constant (Km) and maximum velocity (Vmax) were 8787 μmol/L and 0.86 μmol/L·s, respectively.
Protein purification

Fig. 4 | The activity assay results of tyrosinase TyrVs

a-b. Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from tyrosine to dopaquinone experiments. c-d. Michaelis-Menten plot and Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-DOPA to dopaquinone experiments.