Difference between revisions of "Part:BBa K5398610:Experience"
Line 1: Line 1:
 
===Applications of BBa_K5398601===
 
===Applications of BBa_K5398601===
 
<p>We use the plasmid pET-PC-SUMO-TyrVs to express the tyrosinase TyrVs(BBa_K5398600) which can oxidize tyrosine residues in TRn4-mfp5(BBa_K5398020) to L-DOPA,thereby endowing it with adhesion ability.</p>
 
<p>We use the plasmid pET-PC-SUMO-TyrVs to express the tyrosinase TyrVs(BBa_K5398600) which can oxidize tyrosine residues in TRn4-mfp5(BBa_K5398020) to L-DOPA,thereby endowing it with adhesion ability.</p>
 +
===Characterization===
 +
====Plasmid Construction====
 +
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).
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/sumo-tyrvs.webp" width="400" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 2 | Plasmid pET-PC-SUMO-TyrVs construction results.</b></p>
 +
<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>
 +
    </div>
 +
</body>
 +
</html>
 +
====Protein expression====
 +
a single colony
 +
from a freshly streaked plate of the cells was cultured in 5 mL of LB
 +
medium with 25 μg/mL Ampicillin at 37℃ overnight. The secondary
 +
cultures were prepared with 1% inoculum in 50 mL of LB medium with 25 μg/mL Ampicillin. Cultures were
 +
then incubated at 37℃ and 200 rpm until the optical density at 600 nm
 +
(OD<sub>600</sub>) reached 0.6–0.8. 1 mM IPTG was added to induce production of recombinant proteins and
 +
cultures were further cultivated at 16℃ and 200 rpm for 20 h. The cells
 +
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.
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-pre-expression.webp" width="400" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 3 | Expression of recombinant TyrVs in <i>E. coli</i>BL21 (DE3) with pET-PC-SUMO-TyrVs.</b></p>
 +
<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>
 +
    </div>
 +
</body>
 +
</html>
 +
====Western blotting====
 +
Western bolotting revealed that after induction with IPTG, TyrVs was primarily expressed in its soluble form.
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-western.webp" width="300" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 4 | Western blotting analysis recombinant TyrVs in <i>E. coli</i>BL21 (DE3) with pET-PC-SUMO-TyrVs.</b></p>
 +
<p> Lane 1-3:whole-cell lysate,pellet and supernatant from induced cells with 0.5 mM IPTG respectively.</p>
 +
    </div>
 +
</body>
 +
</html>
 +
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.
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/mizuo-tyrvs.webp" width="400" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 5 | SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.</b></p>
 +
<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>
 +
    </div>
 +
</body>
 +
</html>
 +
====Enzyme activity test====
 +
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.
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-96.webp" width="400" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 6 | The 96 Well Cell Culture Plates of tyrosinase TyrVs.</b></p>
 +
<p>a.The experiment of enzymatic reaction from tyrosine to dopaquinone. b.The experiment of enzymatic reaction from L-DOPA to dopaquinone. </p>
 +
    </div>
 +
</body>
 +
</html>
 +
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.
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/new-abcd.webp" width="400" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 7 | The activity assay results of tyrosinase TyrVs</b></p>
 +
<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>
 +
    </div>
 +
</body>
 +
</html>
 +
====Mathematical modeling Analysis====
 +
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.
 +
<html lang="zh">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
  </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/model.webp" width="400" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 7 | The activity assay results of tyrosinase TyrVs</b></p>
 +
<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>
 +
    </div>
 +
</body>
 +
</html>

Revision as of 03:39, 29 September 2024

Applications of BBa_K5398601

We use the plasmid pET-PC-SUMO-TyrVs to express the tyrosinase TyrVs(BBa_K5398600) which can oxidize tyrosine residues in TRn4-mfp5(BBa_K5398020) to L-DOPA,thereby endowing it with adhesion ability.

Characterization

Plasmid Construction

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 E. coliBL21(DE3).

Protein purification

Fig. 2 | Plasmid pET-PC-SUMO-TyrVs construction results.

a.Expression plasmids of TyrVs. b.PCR results of pET-PC-SUMO-TyrVs. Line 1: Marker. Lines 2-3:Vector;Lines 4-5:Gene.

Protein expression

a single colony from a freshly streaked plate of the cells was cultured in 5 mL of LB medium with 25 μg/mL Ampicillin at 37℃ overnight. The secondary cultures were prepared with 1% inoculum in 50 mL of LB medium with 25 μg/mL Ampicillin. Cultures were then incubated at 37℃ and 200 rpm until the optical density at 600 nm (OD600) reached 0.6–0.8. 1 mM IPTG was added to induce production of recombinant proteins and cultures were further cultivated at 16℃ and 200 rpm for 20 h. The cells 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.

Protein purification

Fig. 3 | Expression of recombinant TyrVs in E. coliBL21 (DE3) with pET-PC-SUMO-TyrVs.

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.

Western blotting

Western bolotting revealed that after induction with IPTG, TyrVs was primarily expressed in its soluble form.

Protein purification

Fig. 4 | Western blotting analysis recombinant TyrVs in E. coliBL21 (DE3) with pET-PC-SUMO-TyrVs.

Lane 1-3:whole-cell lysate,pellet and supernatant from induced cells with 0.5 mM IPTG 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. 5 | 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.

Enzyme activity test

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.

Protein purification

Fig. 6 | The 96 Well Cell Culture Plates of tyrosinase TyrVs.

a.The experiment of enzymatic reaction from tyrosine to dopaquinone. b.The experiment of enzymatic reaction from L-DOPA to dopaquinone.

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-1·s-1, 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-1·s-1, respectively.
Protein purification

Fig. 7 | 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.

Mathematical modeling Analysis

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.

Protein purification

Fig. 7 | The activity assay results of tyrosinase TyrVs

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.