Difference between revisions of "Part:BBa K2215003"
 
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<partinfo>BBa_K2215003 short</partinfo>
 
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Superoxide dismutases (SOD) are the first line of defense against reactive oxygen species when cells become stressed. This makes them important antioxidants in the cell that is responsible for converting superoxide anion radicals into hydrogen peroxide, which can be further converted into water. (Alscher et al., 2002)
 
Superoxide dismutases (SOD) are the first line of defense against reactive oxygen species when cells become stressed. This makes them important antioxidants in the cell that is responsible for converting superoxide anion radicals into hydrogen peroxide, which can be further converted into water. (Alscher et al., 2002)
 
There are different types of SODs in the cell, but this one is known as copper-zinc SOD (CuZn-SOD) that resides in the cytosol, chloroplast, and extracellular space.
 
There are different types of SODs in the cell, but this one is known as copper-zinc SOD (CuZn-SOD) that resides in the cytosol, chloroplast, and extracellular space.
 
This gene was taken from BBa_K1456003 by iGEM14_ATOMS-Turkiye and was codon optimized for Chlamydomonas. As such, this gene is characterized in Chlamydomonas and should be used in Chlamydomonas or should be optimized for the specific species that is to be used.
 
This gene was taken from BBa_K1456003 by iGEM14_ATOMS-Turkiye and was codon optimized for Chlamydomonas. As such, this gene is characterized in Chlamydomonas and should be used in Chlamydomonas or should be optimized for the specific species that is to be used.
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<b style="font-size:1.6rem; text-decoration:underline">Characterization by 2024 iGEM-NJTech-China</b>
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<hr>
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<p>
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A three-point mutation of SOD-1.The 64th base is mutated from phenylalanine to alanine,The 118th base is mutated from valine to alanine and the 61st base is mutated from glycine to alanine,marked by number 15+24+13.
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__TOC__
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==Background==
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<p>
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We obtained the SOD-1 sequence from <a href="https://parts.igem.org/Part:BBa_K2215003">BBa_K2215003</a>.Mutagenesis of specific bases in proteins has proven to be invaluable in probing the contribution of individual amino acid side chains to protein properties. The ability of alanine scanning mutagenesis to provide key biological insights has been demonstrated by early examples, and combinatorial alanine scanning combines the convenience of combinatorial libraries with the insights of site-directed scanning mutagenesis.
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In recent years, methods based on sequence coevolution analysis have shown great potential in enzyme engineering, which can reveal the interactions between amino acid residues and provide a theoretical basis for the modification of enzyme function and structure.
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</P>
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</html>
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==Methods and Results==
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<html>
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So we learned virtual amino acid mutation, used FoldX to perform virtual amino acid mutation based on interaction force on a protein-ligand complex, sorted in descending order according to the relevant change in Gibbs free energy of protein folding (ΔG), identified 28 key amino acids in the active site and amino acid mutation targets that can improve affinity, and tried to improve SOD enzyme activity.
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  .center-img {
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    text-align:center;
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/14huang1.png" alt="28 pairs of mutation sites for virtual amino acid mutation based on interaction forces
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" width="600">
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<p align="center"><b>Figure 2-1</b> 28 pairs of mutation sites for virtual amino acid mutation based on interaction forces</p>
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</div>
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<p>
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We used the scanner to output the final SCI scores of mutations, sorted them in descending order according to the SCI score, and identified the five key amino acids far away from the active site and the amino acid mutation targets that can improve stability, trying to improve the SOD enzyme activity. The SCI index (Sequence Co-evolution Index) is an indicator used in the enzyme activity engineering method based on sequence co-evolution analysis. It is used to evaluate the potential impact of the co-evolution relationship of the enzyme mutation site on the enzyme activity. The calculation of the SCI index takes into account the number and strength of the co-evolutionary relationships between the mutation site and other sites, as well as the distribution of these relationships in the enzyme sequence. The SCI index increases as the frequency of the mutant amino acid pair in the multiple sequence alignment (MSA) increases relative to the wild-type (WT) amino acid pair. Simply put, if a mutation site has a strong co-evolutionary relationship with many other sites in the enzyme sequence, and this relationship is prevalent in the sequence, then the SCI index of this site will be high, indicating that the mutation at this site may have a greater effect on the enzyme activity.</p>
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/14huang2.png" alt="Five pairs of mutation targets for virtual amino acid mutation based on thermal stability and sci
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" width="600">
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<p align="center"><b>Figure 2-2</b> Five pairs of mutation targets for virtual amino acid mutation based on thermal stability and sci</p>
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</div>
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/14bai.png" alt="33 pairs of mutation targets
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" width="600">
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<p align="center"><b>Figure 2-3</b>  33 pairs of mutation targets
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</p>
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</div>
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</html>
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==Iterative mutation==
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1.method
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The activity of SOD was measured using the Beyotime™ SOD enzyme activity kit and A450 was measured using a microplate reader.
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  .center-img {
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    text-align:center;
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  }
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/part1meihuogongshi.png" alt="Calculation formula of SOD inhibition rate and enzyme activity
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" width="600">
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<p align="center"><b>Figure 3-1</b>Calculation formula of SOD inhibition rate and enzyme activity
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</p>
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</div>
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Definition of SOD Enzyme Activity Units: In the aforementioned xanthine oxidase coupled reaction system, when the inhibition percentage reaches 50%, the enzyme activity in the reaction system is defined as one enzyme activity unit (unit).
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</div>
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2.result
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</div>
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（1）single-point mutations of SOD-1
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</div>
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The SOD inhibition rate of the single-point mutations of SOD-1 is shown in the table below.
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  .center-img {
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    text-align:center;
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/sod-inhibition-rate-of-the-1st-mutation.png" alt="SOD inhibition rate of the 1st mutation
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" width="600">
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<p align="center"><b>Figure 3-2</b> SOD inhibition rate of the 1st mutation
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</p>
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</div>
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（2）double-point mutations of SOD-1
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The SOD inhibition rate of the double-point mutations of SOD-1 is shown in the table below.
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<img src="https://static.igem.wiki/teams/5322/wet-lab/sod-inhibition-rate-of-the-2nd-mutation.png" alt=" SOD inhibition rate of the 2nd mutation " width="600">
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<p align="center"><b>Figure 3-3</b>  SOD inhibition rate of the 2nd mutation
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</p>
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</div>
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（3）three-point mutations of SOD-1
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The SOD inhibition rate of the three-point mutations of SOD-1 is shown in the table below.
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At the seven mutation sites 2, 13, 15, 24, 26, 30, and 33, we generated 35 mutation combinations using permutation and combination methods. Considering cost and time constraints, we selected four optimal pairs of mutations—13+30, 2+33, 15+24, and 26+33—based on the results from the double mutations as templates, resulting in the following 20 combinations:
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/20-groups-of-three-point-mutations.png" alt="20 groups of three-point mutations
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" width="600">
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<p align="center"><b>Figure 3-4</b>20 groups of three-point mutations
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</p>
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</div>
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The SOD inhibition rate of the double-point mutations of SOD-1 is shown in the table below.
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<style>
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  .center-img {
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    text-align:center;
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  }
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</style>
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/sod-inhibition-rate-of-the-3rd-mutation.png" alt="SOD inhibition rate of the 3rd mutation
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" width="600">
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<p align="center"><b>Figure 3-5</b>SOD inhibition rate of the 3rd mutation
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</p>
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</div>
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</html>
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==Functional Expression==
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<html>
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An inducible SOD-1 expression plasmid was constructed by three-point mutation of SOD-1.The 64th base is mutated from phenylalanine to alanine,The 118th base is mutated from valine to alanine and the 61st base is mutated from glycine to alanine,marked by number 15+24+13.We took 2 mL of the overnight induced bacterial culture and used the Beyotime™ bacterial active protein extraction reagent to extract the protein. SOD activity was measured using the Beyotime™ SOD activity detection kit.
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<style>
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  .center-img {
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    text-align:center;
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  }
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</style>
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/33333333.png" alt="Protein standard curve
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" width="600">
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<p align="center"><b>Figure 4-1</b> Protein standard curve
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</p>
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</div>
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The original data are shown in the table below, SOD Plus has an inhibition rate of 58.57% and an enzyme activity of 1.4136 U, which represents an increase of 0.5399 U over the unmutated SOD.
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<style>
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  .center-img {
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    text-align:center;
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  }
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</style>
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/12-13.png" alt="Calculation formula of SOD inhibition rate and enzyme activity
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" width="600">
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<p align="center"><b>Figure 4-2</b>Data of the Inhibition rate and enzyme activity
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</p>
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</div>
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<div class="center-img">
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<img src="https://static.igem.wiki/teams/5322/wet-lab/sodplus-15-24-13.png" alt="Comparison of inhibition rate and enzyme activity
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" width="600">
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<p align="center"><b>Figure 4-3</b>Comparison of inhibition rate and enzyme activity
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</p>
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</div>
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</html>
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==References==
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<html>
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1.Combinatorial alanine-scanning Kim L Morrison and Gregory A Weiss.Department of Chemistry, University of California, Irvine,CA 92697-2025, USA.
 +
 +
2.Kim,D. , Noh,M.H. , Park,M. , Kim,I. , Ahn,H. , Ye,D. , Jung,G.Y. ,& Kim , S.(2022).Enzyme activity engineering based on sequence co-evolution analysis.Metabolic Engineering,74(2),49-60.https://doi.org/10.1016/j.ymben.2022.09.001
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 +
3.4.Ge Qu, Jing Zhao, elt. Recent advances in directed evolution. Chinese Journal of Biotechnology. Jan. 25, 2018, 34(1): 1−11
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</html>
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2215003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2215003 SequenceAndFeatures</partinfo>

Latest revision as of 13:10, 2 October 2024


CuZn SOD

Superoxide dismutases (SOD) are the first line of defense against reactive oxygen species when cells become stressed. This makes them important antioxidants in the cell that is responsible for converting superoxide anion radicals into hydrogen peroxide, which can be further converted into water. (Alscher et al., 2002) There are different types of SODs in the cell, but this one is known as copper-zinc SOD (CuZn-SOD) that resides in the cytosol, chloroplast, and extracellular space. This gene was taken from BBa_K1456003 by iGEM14_ATOMS-Turkiye and was codon optimized for Chlamydomonas. As such, this gene is characterized in Chlamydomonas and should be used in Chlamydomonas or should be optimized for the specific species that is to be used.

Characterization by 2024 iGEM-NJTech-China

A three-point mutation of SOD-1.The 64th base is mutated from phenylalanine to alanine,The 118th base is mutated from valine to alanine and the 61st base is mutated from glycine to alanine,marked by number 15+24+13.

Background

We obtained the SOD-1 sequence from BBa_K2215003.Mutagenesis of specific bases in proteins has proven to be invaluable in probing the contribution of individual amino acid side chains to protein properties. The ability of alanine scanning mutagenesis to provide key biological insights has been demonstrated by early examples, and combinatorial alanine scanning combines the convenience of combinatorial libraries with the insights of site-directed scanning mutagenesis. In recent years, methods based on sequence coevolution analysis have shown great potential in enzyme engineering, which can reveal the interactions between amino acid residues and provide a theoretical basis for the modification of enzyme function and structure.

Methods and Results

So we learned virtual amino acid mutation, used FoldX to perform virtual amino acid mutation based on interaction force on a protein-ligand complex, sorted in descending order according to the relevant change in Gibbs free energy of protein folding (ΔG), identified 28 key amino acids in the active site and amino acid mutation targets that can improve affinity, and tried to improve SOD enzyme activity.

28 pairs of mutation sites for virtual amino acid mutation based on interaction forces

Figure 2-1 28 pairs of mutation sites for virtual amino acid mutation based on interaction forces

We used the scanner to output the final SCI scores of mutations, sorted them in descending order according to the SCI score, and identified the five key amino acids far away from the active site and the amino acid mutation targets that can improve stability, trying to improve the SOD enzyme activity. The SCI index (Sequence Co-evolution Index) is an indicator used in the enzyme activity engineering method based on sequence co-evolution analysis. It is used to evaluate the potential impact of the co-evolution relationship of the enzyme mutation site on the enzyme activity. The calculation of the SCI index takes into account the number and strength of the co-evolutionary relationships between the mutation site and other sites, as well as the distribution of these relationships in the enzyme sequence. The SCI index increases as the frequency of the mutant amino acid pair in the multiple sequence alignment (MSA) increases relative to the wild-type (WT) amino acid pair. Simply put, if a mutation site has a strong co-evolutionary relationship with many other sites in the enzyme sequence, and this relationship is prevalent in the sequence, then the SCI index of this site will be high, indicating that the mutation at this site may have a greater effect on the enzyme activity.

Five pairs of mutation targets for virtual amino acid mutation based on thermal stability and sci

Figure 2-2 Five pairs of mutation targets for virtual amino acid mutation based on thermal stability and sci

33 pairs of mutation targets

Figure 2-3 33 pairs of mutation targets

Iterative mutation

1.method The activity of SOD was measured using the Beyotime™ SOD enzyme activity kit and A450 was measured using a microplate reader.

Calculation formula of SOD inhibition rate and enzyme activity

Figure 3-1Calculation formula of SOD inhibition rate and enzyme activity

Definition of SOD Enzyme Activity Units: In the aforementioned xanthine oxidase coupled reaction system, when the inhibition percentage reaches 50%, the enzyme activity in the reaction system is defined as one enzyme activity unit (unit).
2.result
（1）single-point mutations of SOD-1
The SOD inhibition rate of the single-point mutations of SOD-1 is shown in the table below.
SOD inhibition rate of the 1st mutation

Figure 3-2 SOD inhibition rate of the 1st mutation

（2）double-point mutations of SOD-1 The SOD inhibition rate of the double-point mutations of SOD-1 is shown in the table below.
SOD inhibition rate of the 2nd mutation

Figure 3-3 SOD inhibition rate of the 2nd mutation

（3）three-point mutations of SOD-1 The SOD inhibition rate of the three-point mutations of SOD-1 is shown in the table below. At the seven mutation sites 2, 13, 15, 24, 26, 30, and 33, we generated 35 mutation combinations using permutation and combination methods. Considering cost and time constraints, we selected four optimal pairs of mutations—13+30, 2+33, 15+24, and 26+33—based on the results from the double mutations as templates, resulting in the following 20 combinations:
20 groups of three-point mutations

Figure 3-420 groups of three-point mutations

The SOD inhibition rate of the double-point mutations of SOD-1 is shown in the table below.
SOD inhibition rate of the 3rd mutation

Figure 3-5SOD inhibition rate of the 3rd mutation

Functional Expression

An inducible SOD-1 expression plasmid was constructed by three-point mutation of SOD-1.The 64th base is mutated from phenylalanine to alanine,The 118th base is mutated from valine to alanine and the 61st base is mutated from glycine to alanine,marked by number 15+24+13.We took 2 mL of the overnight induced bacterial culture and used the Beyotime™ bacterial active protein extraction reagent to extract the protein. SOD activity was measured using the Beyotime™ SOD activity detection kit.

Protein standard curve

Figure 4-1 Protein standard curve

The original data are shown in the table below, SOD Plus has an inhibition rate of 58.57% and an enzyme activity of 1.4136 U, which represents an increase of 0.5399 U over the unmutated SOD.
Calculation formula of SOD inhibition rate and enzyme activity

Figure 4-2Data of the Inhibition rate and enzyme activity

Comparison of inhibition rate and enzyme activity

Figure 4-3Comparison of inhibition rate and enzyme activity

References

1.Combinatorial alanine-scanning Kim L Morrison and Gregory A Weiss.Department of Chemistry, University of California, Irvine,CA 92697-2025, USA. 2.Kim,D. , Noh,M.H. , Park,M. , Kim,I. , Ahn,H. , Ye,D. , Jung,G.Y. ,& Kim , S.(2022).Enzyme activity engineering based on sequence co-evolution analysis.Metabolic Engineering,74(2),49-60.https://doi.org/10.1016/j.ymben.2022.09.001 3.4.Ge Qu, Jing Zhao, elt. Recent advances in directed evolution. Chinese Journal of Biotechnology. Jan. 25, 2018, 34(1): 1−11


Sequence and Features


Assembly Compatibility:
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