Difference between revisions of "Part:BBa K4763007"

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         <img src="https://static.igem.wiki/teams/4763/wiki/atpcs-part-igem-registry/atpcs-predicted-structure.png" width="250">
 
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         <figcaption><b>Figure 2.</b> Predicted structure with the best PAE obtained from ColabFold showing the modeled AtPCS sequence.</figcaption>
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         <figcaption><b>Figure 2.</b> Predicted structure with the best PAE obtained from ColabFold showing the modeled <i>At</i>PCS sequence.</figcaption>
 
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MD simulations were performed to assess if the predicted structure obtained of the system was conserved after including biophysical potentials. A preliminary simulation was run with GROMACS using model 5 from ColabFold. The topology was generated with the force field AMBER99SB-ILDN and the water model TIP3P (<b>Figure 3</b>).
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        <img src="https://static.igem.wiki/teams/4763/wiki/atpcs-part-igem-registry/atpcs-trajectory.gif" width="350">
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        <figcaption><b>Figure 3.</b> Trajectory (100 ps frame<sup>-1</sup>) of <i>At</i>PCS compared to predicted ColabFold (gray) structure.</figcaption>
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==Cloning <i>At</i>PCS insert into pET28(+) vector==
 
==Cloning <i>At</i>PCS insert into pET28(+) vector==
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The gene sequence for <i>At</i>PCS was transformed into the pET28b(+) vector. This was done using T4 DNA Ligase (New England Biolabs). Table X shows the components used for the ligation reaction at a 1:5 molar ratio:  
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The gene sequence for <i>At</i>PCS was transformed into the pET28b(+) vector. This was done using T4 DNA Ligase (New England Biolabs). <b>Table 1</b> shows the components used for the ligation reaction at a 1:7 molar ratio:  
  
 
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!Reagent !! Quantity
 
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| style="text-align:center;" style="width: 80%;" | Insert || 192 ng
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| style="text-align:center;" style="width: 80%;" | Insert || 192 ng (4.9 µL)
 
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| style="text-align:center;" style="width: 80%;" | T4 DNA ligase buffer || 2 µL
 
| style="text-align:center;" style="width: 80%;" | T4 DNA ligase buffer || 2 µL
 
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| style="text-align:center;" style="width: 80%;" | Vector || 100 ng
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| style="text-align:center;" style="width: 80%;" | Vector || 100 ng (2.6 µL)
 
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| style="text-align:center;" style="width: 80%;" | T4 DNA ligase|| 1 µL
 
| style="text-align:center;" style="width: 80%;" | T4 DNA ligase|| 1 µL
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The ligation was then transformed into <i>E. coli</i> TOP10 by adding 5 μL of the ligation reaction to 50 μL of competent cells following the heat shock method. After incubation colonies were observed indicating successful transformation (<b>Figure 4</b>).
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The ligation was then transformed into <i>E. coli</i> TOP10 by adding 10 μL of the ligation reaction to 50 μL of competent cells following the heat shock method. After incubation colonies were observed indicating successful transformation (<b>Figure 4</b>).
  
 
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         <figcaption><b>Figure 4.</b> Transformation of pET28b(+)_<i>At</i>PCS plasmid into <i>E. coli</i> TOP10 cells. A) 1 hour ligation. B) Overnight ligation.</figcaption>
 
         <figcaption><b>Figure 4.</b> Transformation of pET28b(+)_<i>At</i>PCS plasmid into <i>E. coli</i> TOP10 cells. A) 1 hour ligation. B) Overnight ligation.</figcaption>
 
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==Screening of transformed cells by colony PCR==
 
==Screening of transformed cells by colony PCR==
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==Restriction enzyme digestion test==
 
==Restriction enzyme digestion test==
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To identify, through an additional methodology, the clones that were correctly transformed with the gene, restriction assays were performed on plasmids obtained from colonies 1-3 using the NdeI and EcoRI-HF enzymes. For both vector and insert, DNA concentration was stated as 500 nanograms. <b>Table 2</b> displays the protocol followed for a 50 µL reaction.
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To identify, through an additional methodology, the clones that were correctly transformed with the gene, restriction assays were performed on plasmids obtained from colonies 1-3 using the NdeI and EcoRI-HF enzymes. <b>Table 2</b> displays the protocol followed for a 50 µL reaction.
  
 
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| style="text-align:center;" style="width: 80%;" | Nuclease-free water || add to 50 µL
 
| style="text-align:center;" style="width: 80%;" | Nuclease-free water || add to 50 µL
 
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| style="text-align:center;" style="width: 80%;" | 10X NEBuffer || 5 µL
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| style="text-align:center;" style="width: 80%;" | rCutSmart Buffer || 5 µL
 
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| style="text-align:center;" style="width: 80%;" | <i>At</i>PCS_pET28b(+) candidates || up to 500 ng
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| style="text-align:center;" style="width: 80%;" | <i>At</i>PCS_pET28b(+) candidates || 400 ng (10 µL)
 
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| style="text-align:center;" style="width: 80%;" | <i>NdeI</i> restriction enzyme || 1 µL
 
| style="text-align:center;" style="width: 80%;" | <i>NdeI</i> restriction enzyme || 1 µL
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==Protein overexpression trials in <i>E. coli</i> BL21==
 
==Protein overexpression trials in <i>E. coli</i> BL21==
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Overexpression trials were performed by induction with 1M isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16°C overnight. Cell lysis was performed through sonication and intracellular protein presence and concentration was measured through an SDS-PAGE gel, as shown in <b>Figure 7</b>. The gel showed a very faint band present at the appropriate weight for the <i>At</i>PCS gene.
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Overexpression trials were performed by induction with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16°C overnight. Cell lysis was performed through sonication and intracellular protein presence and concentration was measured through an SDS-PAGE gel, as shown in <b>Figure 7</b>. The gel showed a very faint band present at the appropriate weight for the <i>At</i>PCS gene.
  
 
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[3]. Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., & Steinegger, M. (2022). ColabFold: making protein folding accessible to all. <i>Nature methods, 19</i>(6), 679–682. https://doi.org/10.1038/s41592-022-01488-1
 
[3]. Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., & Steinegger, M. (2022). ColabFold: making protein folding accessible to all. <i>Nature methods, 19</i>(6), 679–682. https://doi.org/10.1038/s41592-022-01488-1
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Latest revision as of 05:17, 12 October 2023


AtPCS (phytochelatin synthase) coding sequence

Phytochelatin synthase (AtPCS) coding sequence from Arabidopsis thaliana. This enzyme catalyzes the final step in the biosynthesis of phytochelatins (PCs) using as a co-substrate the toxic heavy metal cadmium.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 649
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 649
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 649
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

The enzyme chosen for the generation of the biopart was phytochelatin synthase or PCS (EC 2.3.2.15), which catalyzes the synthesis of glutathione (GSH) polymers called phytochelatins (PCs) in presence of toxic heavy metals (Figure 1). These are produced as a mechanism of resistance and accumulation in algae, yeast, plants, and worms in response to cadmium stress, mainly. Phytochelatins bind to metals intracellularly and render them inactive. This is because the substrate of PCS is GSH (as aforementioned) and other metal-bis-glutathione complexes. Additionally, the enzyme activity requires the presence of specific heavy metals (depending on the species under study), with cadmium being the main inducer in plants and other organisms. The complexes formed by the metal and PCs can be subsequently compartmentalized into vacuoles, chloroplasts, and mitochondria (García-García et al., 2014; 2020).

The PCS enzyme from the model organism Arabidopsis thaliana (AtPCS1) was selected for the creation of this biopart due to its more extensive characterization in the synthesis of phytochelatins.


Figure 1. Phytochelatin synthesis from GSH, catalyzed by PCS. .


Characterization

Molecular dynamics

A predicted structure was obtained through ColabFold (Mirdita et al., 2022) using existing crystallographic structures for each constituent protein (Figure 2). To lend more confidence to the predicted structure, molecular dynamics (MD) simulations of the obtained model were performed. This helped assess if the predicted structure was maintained after the protein was exposed to biophysical potentials.

Figure 2. Predicted structure with the best PAE obtained from ColabFold showing the modeled AtPCS sequence.

MD simulations were performed to assess if the predicted structure obtained of the system was conserved after including biophysical potentials. A preliminary simulation was run with GROMACS using model 5 from ColabFold. The topology was generated with the force field AMBER99SB-ILDN and the water model TIP3P (Figure 3).

Figure 3. Trajectory (100 ps frame-1) of AtPCS compared to predicted ColabFold (gray) structure.

Cloning AtPCS insert into pET28(+) vector

The gene sequence for AtPCS was transformed into the pET28b(+) vector. This was done using T4 DNA Ligase (New England Biolabs). Table 1 shows the components used for the ligation reaction at a 1:7 molar ratio:

Table 1. 20 μL ligation of AtPCS insert and pET28b(+) vector.
Reagent Quantity
Insert 192 ng (4.9 µL)
T4 DNA ligase buffer 2 µL
Vector 100 ng (2.6 µL)
T4 DNA ligase 1 µL
Nuclease-free water 9.5 µL

The ligation was then transformed into E. coli TOP10 by adding 10 μL of the ligation reaction to 50 μL of competent cells following the heat shock method. After incubation colonies were observed indicating successful transformation (Figure 4).

Figure 4. Transformation of pET28b(+)_AtPCS plasmid into E. coli TOP10 cells. A) 1 hour ligation. B) Overnight ligation.

Screening of transformed cells by colony PCR

To verify the presence of AtPCS gene in the pET28b(+) plasmids, a colony PCR was performed with five transformed colonies, where a band was observed near the 1500 bp mark, which matches the length of the gene (1461 bp) (Figure 5).

Figure 5. Amplification of AtPCS gene from random colonies corresponding to transformation of 1 hour ligation into E. coli TOP10 cells. The ladder used was 1kb Plus ladder from Invitrogen. Colonies were randomly selected and numbered from 1 to 5. A band is observed near the 1500 bp mark, which matches the length of the gene (1461 bp).


Restriction enzyme digestion test

To identify, through an additional methodology, the clones that were correctly transformed with the gene, restriction assays were performed on plasmids obtained from colonies 1-3 using the NdeI and EcoRI-HF enzymes. Table 2 displays the protocol followed for a 50 µL reaction.

Table 2. Restriction digest conditions.
Reagent Quantity
Nuclease-free water add to 50 µL
rCutSmart Buffer 5 µL
AtPCS_pET28b(+) candidates 400 ng (10 µL)
NdeI restriction enzyme 1 µL
EcoRI restriction enzyme 1 µL

Figure 6 shows the agarose gel with the results of the enzymatic digestions, where a band corresponding to the length of the AtPCS gene was identified in sample D1 from colony 1.

Figure 6. Digestion of pET28b(+) plasmid with NdeI and EcoRI-HF restriction enzymes. The ladder used was 1kb Plus ladder from Invitrogen. A positive result for AtPCS is observed in colony 1 lane D1.

Protein overexpression trials in E. coli BL21

Overexpression trials were performed by induction with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16°C overnight. Cell lysis was performed through sonication and intracellular protein presence and concentration was measured through an SDS-PAGE gel, as shown in Figure 7. The gel showed a very faint band present at the appropriate weight for the AtPCS gene.

Figure 7. SDS-PAGE gel showing protein overexpression results for induced colonies. In wells corresponding to colonies 4-7 supernatants, a very faint blurred band near to 55 kDa (AtPCS molecular weight) is visible, contrary to negative control.

References

[1]. García-García, J. D., Sánchez-Thomas, R., Saavedra, E., Fernández-Velasco, D. A., Romero-Romero, S., Casanova-Figueroa, K. I., … Moreno-Sánchez, R. (2020). Mapping the metal-catalytic site of a zinc-activated phytochelatin synthase. Algal Research, 101890. doi:10.1016/j.algal.2020.101890

[2]. García-García, J. D., Girard, L., Hernández, G., Saavedra, E., Pardo, J. P., Rodríguez-Zavala, J. S., Encalada, R., Reyes-Prieto, A., Mendoza-Cózatl, D. G., & Moreno-Sánchez, R. (2014). Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis. Metallomics : integrated biometal science, 6(3), 604–616. https://doi.org/10.1039/c3mt00313b

[3]. Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., & Steinegger, M. (2022). ColabFold: making protein folding accessible to all. Nature methods, 19(6), 679–682. https://doi.org/10.1038/s41592-022-01488-1