Difference between revisions of "Part:BBa K5439001"

(→‎Cloning TjPCs insert into pET28b(+) vector)
(→‎Cloning TjPCs insert into pET28b(+) vector)
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=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.
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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 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
 
!Reagent !! Volume (µL) 3:1 ratio !! Volume (µL) 5:1 ratio
 
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|-
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| style="text-align:center;" style="width: 60%;" | pet28b(+) || 6.7 µL || 6.7 µL
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| style="text-align:center;" style="width: 80%;" | pet28b(+) || 6.7 µL
 
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|-
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| style="text-align:center;" style="width: 60%;" | TjPCs || 5.8 µL || 9.7 µL
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| style="text-align:center;" style="width: 80%;" | TjPCs || 9.7 µL
 
|--
 
|--
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| style="text-align:center;" style="width: 60%;" | T4 DNA Ligase Buffer || 2 µL || 2 µL
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| style="text-align:center;" style="width: 80%;" | T4 DNA Ligase Buffer 2 µL
 
|-
 
|-
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| style="text-align:center;" style="width: 60%;" | T4 DNA ligase|| 0.2 µL || 0.2 µL
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| style="text-align:center;" style="width: 80%;" | T4 DNA ligase|| 0.2 µL
 
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|-
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| style="text-align:center;" style="width: 60%;" | Nuclease-free water|| 5.3 µL || 1.4 µL
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| style="text-align:center;" style="width: 80%;" | Nuclease-free water|| 1.4 µL
 
|}
 
|}
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After 1 hour of incubation at 22 ºC, the resulting ligation was transformed through heat shock in E.coli BL21 chemically competent cells. The results from the transformation can be noted in <b>Figure 1</b>.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 22:58, 1 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 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 PCs from Thlaspi japonicum (TjPCs) provides cadmium tolerance when it is heterologous expressed in Saccharomyces cerevisiae, making the synthesis of this enzyme of interest for Cd pollution problems (Mizuno et al., 2003).

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 with 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 Volume (µL) 3:1 ratio Volume (µL) 5:1 ratio
pet28b(+) 6.7 µL
TjPCs 9.7 µL
T4 DNA Ligase Buffer 2 µL
T4 DNA ligase 0.2 µL
Nuclease-free water 1.4 µL

After 1 hour of incubation at 22 ºC, the resulting ligation was transformed through heat shock in E.coli BL21 chemically competent cells. The results from the transformation can be noted in Figure 1.