Difference between revisions of "Part:BBa K5439001"

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=Usage and Biology=
 
=Usage and Biology=
  
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The enzyme chosen for the biopart was phytochelatin synthase (EC:2.3.2.15) as a detector for the presence of cadmium. This enzyme catalyzes the synthesis of glutathione (GSH) polymers, or phytochelatins (PCs). These molecules are the most studied chelators for the detoxification of heavy metals in plants, and they serve as high-affinity chelators for the detoxification of heavy metals such as cadmium, zinc, and nickel. PCs bind to these metals through their thiol groups and inactivate them, storing the PC-metal complex in the cytosol (in the case of plants) or in chloroplasts (in the case of algae or protists) (Rea, 2012; García-García, 2014).
+
The enzyme chosen for the biopart was phytochelatin synthase (EC:2.3.2.15) as a detector for the presence of cadmium. This enzyme catalyzes an enzymatic reaction from glutathione tripeptide (GSH) that synthesizes phytochelatins (PCs). PCs are high cysteine polypeptides involved in heavy metal chelation, used by hyperaccumulating plants as the main mechanism in the detoxification of metals such as cadmium, zinc, and nickel (Faizan et al., 2024). In order to produce PCs, phytochelatin synthesase genes are expressed by the presence of ions from high concentrations of heavy metals. The process occurs by transferring γ-Glu-Cys from a donor molecule (glutathione) to an aceptor molecule (phytochelatin synthesase), which catalyzes the synthesis of PCs. Subsequently, PCs binds heavy metals ions by thiol groups, which then transports them to the vacuoles where they present minor damage for plants (Zitka et al., 2011)
  
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The PCs from <i>Thlaspi japonicum</i> (TjPCs) provides cadmium tolerance when it is heterologous expressed in <i>Saccharomyces cerevisiae</i>, making the synthesis of this enzyme of interest for Cd pollution problems (Mizuno et al., 2003).  
+
The PCs from <i>Thlaspi japonicum</i> (TjPCs) was selected as it provides cadmium tolerance when it is heterologously expressed in <i>Saccharomyces cerevisiae</i>, making the synthesis of this enzyme of interest for Cd pollution problems (Mizuno et al., 2011).
 +
 
 +
=Characterization=
 +
Using the coding sequence for the protein, ColabFold (Jumper et al., 2021) was used in order to obtain a prediction of the structure, considering the best Predicted Aligned Error (PAE) (<b>Figure 1</b>).
 +
 
 +
<html>
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<head>
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<style>
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    figure {
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        text-align: center;
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    }
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    figcaption {
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        font-size: 12px;
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    }
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</style>
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</head>
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<body>
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    <figure>
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        <img src="https://static.igem.wiki/teams/5439/tjpcs-solo.png" width="600">
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        <figcaption><b>Figure 1.</b> Predicted structure with the best PAE obtained from ColabFold showing the modeled TjPCs sequence.</figcaption>
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    </figure>
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</body>
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</html>
  
 
=Cloning TjPCs insert into pET28b(+) vector=
 
=Cloning TjPCs insert into pET28b(+) vector=
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In order heterologously overexpress PCs in <i>Escherichia coli</i>, a ligation was carried out with TjPCs and a vector pET28b(+). This was achieved with T4 DNA ligase (Invitrogen), with molar ratios 3:1 and 5:1 following the protocol obseved in Table 1.
+
In order heterologously overexpress PCs in <i>Escherichia coli</i>, a ligation was carried out with TjPCs and a vector pET28b(+). This was achieved utilizing T4 DNA ligase (Invitrogen), with 5:1 molar ratio following the protocol as observed in <b>Table 1</b>.
  
 
{| class="wikitable" style="margin:auto; text-align:center; length: 80%"
 
{| class="wikitable" style="margin:auto; text-align:center; length: 80%"
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|+ Table 1. Ligation of TjPCs insert and pET28b(+) vector (3:1 and 5:1 molar ratios).
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|+ Table 1. Ligation of TjPCs insert and pET28b(+) vector (5:1 molar ratio).
 
|-
 
|-
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!Reagent !! Volume (µL) 3:1 ratio !! Volume (µL) 5:1 ratio
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!Reagent !! Quantity (5:1 ratio)
 
|-
 
|-
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| style="text-align:center;" style="width: 80%;" | pet28b(+) || 6.7 µL || 6.7 µL
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| style="text-align:center;" style="width: 60%;" |pet28b(+)||50 ng
 
|-
 
|-
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| style="text-align:center;" style="width: 80%;" | TjPCs || 5.8 µL || 9.7 µL
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| style="text-align:center;" style="width: 60%;" |TjPCs||5:1 molar ratio over vector
 
|--
 
|--
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| style="text-align:center;" style="width: 80%;" | T4 DNA Ligase Buffer || 2 µL || 2 µL
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| style="text-align:center;" style="width: 60%;" |T4 DNA Ligase Buffer||2 µL
 
|-
 
|-
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| style="text-align:center;" style="width: 80%;" | T4 DNA ligase|| 0.2 µL || 0.2 µL
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| style="text-align:center;" style="width: 60%;" |T4 DNA ligase||1 Weiss U
 
|-
 
|-
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| style="text-align:center;" style="width: 80%;" | Nuclease-free water|| 5.3 µL || 1.4 µL
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| style="text-align:center;" style="width: 60%;" |Nuclease-free water||To 20 µL
 
|}
 
|}
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 +
After 1 hour incubation at 22 ºC, 5 µL of the resulting ligation were transformed through heat shock in 50 µL of <i>E. coli</i> BL21 chemically competent cells. The successful results from the transformation can be noted in <b>Figure 2</b>, incubated overnight at 37 ºC in LB agar and kanamycin (50 μg/mL).
 +
 +
<html>
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<head>
 +
<style>
 +
    figure {
 +
        text-align: center;
 +
    }
 +
 +
    figcaption {
 +
        font-size: 12px;
 +
    }
 +
 +
</style>
 +
</head>
 +
<body>
 +
    <figure>
 +
        <img src="https://static.igem.wiki/teams/5439/pet28b-tjpcs-ligation.png" width="600">
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        <figcaption><b>Figure 2.</b> Transformation of pET28b(+)_TjPCs plasmid into <i>E. coli</i> BL21 cells.</figcaption>
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    </figure>
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</body>
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</html>
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=References=
 +
Faizan, M., Alam, P., Hussain, A., Karabulut, F., Tonny, S. H., Cheng, S. H., Yusuf, M., Adil, M. F., Sehar, S., Alomrani, S. O., Albalawi, T., & Hayat, S. (2024). Phytochelatins: Key regulator against heavy metal toxicity in plants. Plant Stress, 11, 100355. https://doi.org/10.1016/j.stress.2024.100355
 +
Zitka, O., Krystofova, O., Sobrova, P., Adam, V., Zehnalek, J., Beklova, M., & Kizek, R. (2011). Phytochelatin synthase activity as a marker of metal pollution. Journal of Hazardous Materials, 192(2), 794–800. https://doi.org/10.1016/j.jhazmat.2011.05.088
 +
 +
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., Bridgland, A., Meyer, C., Kohl, S. A. A., Ballard, A. J., Cowie, A., Romera-Paredes, B., Nikolov, S., Jain, R., Adler, J., … Hassabis, D. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 596(7873), 583–589. https://doi.org/10.1038/s41586-021-03819-2
 +
 +
Mizuno, T., Sonoda, T., Horie, K., Senoo, K., & Tanaka, A. (2011, November 22). Cloning and characterization of phytochelatin synthase from a Nickel hyperaccumulator Thlaspi japonicum and its expression in yeast. Soil Science and Plant Nutrition. https://doi.org/10.1080/00380768.2003.10410009
 +
 +
Zitka, O., Krystofova, O., Sobrova, P., Adam, V., Zehnalek, J., Beklova, M., & Kizek, R. (2011). Phytochelatin synthase activity as a marker of metal pollution. Journal of Hazardous Materials, 192(2), 794–800. https://doi.org/10.1016/j.jhazmat.2011.05.088
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 03:49, 2 October 2024


TjPCs (phytochelatin synthase) coding sequence

Phytochelatin synthase coding sequence from Thlaspi japonicum. This gluthanione-γ-glutamylcysteinyltransferase posttranslationally synthesizes phytochelatins in the presence of heavy metals and gluthanione as a mechanism of heavy metal detoxification.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 181
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 31
    Illegal BglII site found at 1440
    Illegal XhoI site found at 1462
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

The enzyme chosen for the biopart was phytochelatin synthase (EC:2.3.2.15) as a detector for the presence of cadmium. This enzyme catalyzes an enzymatic reaction from glutathione tripeptide (GSH) that synthesizes phytochelatins (PCs). PCs are high cysteine polypeptides involved in heavy metal chelation, used by hyperaccumulating plants as the main mechanism in the detoxification of metals such as cadmium, zinc, and nickel (Faizan et al., 2024). In order to produce PCs, phytochelatin synthesase genes are expressed by the presence of ions from high concentrations of heavy metals. The process occurs by transferring γ-Glu-Cys from a donor molecule (glutathione) to an aceptor molecule (phytochelatin synthesase), which catalyzes the synthesis of PCs. Subsequently, PCs binds heavy metals ions by thiol groups, which then transports them to the vacuoles where they present minor damage for plants (Zitka et al., 2011)

The PCs from Thlaspi japonicum (TjPCs) was selected as it provides cadmium tolerance when it is heterologously expressed in Saccharomyces cerevisiae, making the synthesis of this enzyme of interest for Cd pollution problems (Mizuno et al., 2011).

Characterization

Using the coding sequence for the protein, ColabFold (Jumper et al., 2021) was used in order to obtain a prediction of the structure, considering the best Predicted Aligned Error (PAE) (Figure 1).

Figure 1. Predicted structure with the best PAE obtained from ColabFold showing the modeled TjPCs sequence.

Cloning TjPCs insert into pET28b(+) vector

In order heterologously overexpress PCs in Escherichia coli, a ligation was carried out with TjPCs and a vector pET28b(+). This was achieved utilizing T4 DNA ligase (Invitrogen), with 5:1 molar ratio following the protocol as observed in Table 1.

Table 1. Ligation of TjPCs insert and pET28b(+) vector (5:1 molar ratio).
Reagent Quantity (5:1 ratio)
pet28b(+) 50 ng
TjPCs 5:1 molar ratio over vector
T4 DNA Ligase Buffer 2 µL
T4 DNA ligase 1 Weiss U
Nuclease-free water To 20 µL

After 1 hour incubation at 22 ºC, 5 µL of the resulting ligation were transformed through heat shock in 50 µL of E. coli BL21 chemically competent cells. The successful results from the transformation can be noted in Figure 2, incubated overnight at 37 ºC in LB agar and kanamycin (50 μg/mL).

Figure 2. Transformation of pET28b(+)_TjPCs plasmid into E. coli BL21 cells.

References

Faizan, M., Alam, P., Hussain, A., Karabulut, F., Tonny, S. H., Cheng, S. H., Yusuf, M., Adil, M. F., Sehar, S., Alomrani, S. O., Albalawi, T., & Hayat, S. (2024). Phytochelatins: Key regulator against heavy metal toxicity in plants. Plant Stress, 11, 100355. https://doi.org/10.1016/j.stress.2024.100355 Zitka, O., Krystofova, O., Sobrova, P., Adam, V., Zehnalek, J., Beklova, M., & Kizek, R. (2011). Phytochelatin synthase activity as a marker of metal pollution. Journal of Hazardous Materials, 192(2), 794–800. https://doi.org/10.1016/j.jhazmat.2011.05.088

Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., Bridgland, A., Meyer, C., Kohl, S. A. A., Ballard, A. J., Cowie, A., Romera-Paredes, B., Nikolov, S., Jain, R., Adler, J., … Hassabis, D. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 596(7873), 583–589. https://doi.org/10.1038/s41586-021-03819-2

Mizuno, T., Sonoda, T., Horie, K., Senoo, K., & Tanaka, A. (2011, November 22). Cloning and characterization of phytochelatin synthase from a Nickel hyperaccumulator Thlaspi japonicum and its expression in yeast. Soil Science and Plant Nutrition. https://doi.org/10.1080/00380768.2003.10410009

Zitka, O., Krystofova, O., Sobrova, P., Adam, V., Zehnalek, J., Beklova, M., & Kizek, R. (2011). Phytochelatin synthase activity as a marker of metal pollution. Journal of Hazardous Materials, 192(2), 794–800. https://doi.org/10.1016/j.jhazmat.2011.05.088