Difference between revisions of "Part:BBa K5108002"
 
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<partinfo>BBa_K5108002 short</partinfo>
 
<partinfo>BBa_K5108002 short</partinfo>
  
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<i>P. fluorescens</i> catalase KatB ORF
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Catalse from <i>P. fluorescens</i> SBW25
  
 
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     <li style="color: blue;">Usage and Biology</li>
 
     <li style="color: blue;">Usage and Biology</li>
 
<li style="color: blue;">Sequence and Features</li>
 
<li style="color: blue;">Sequence and Features</li>
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<li style="color: blue;">Modeling</li>
 
 
     <li style="color: blue;">Characterization and Measurements
 
     <li style="color: blue;">Characterization and Measurements
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        <ol>
 
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            <li style="color: blue;">SDS-PAGE</li>
 
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            <li style="color: blue;">Growth analysis</li>
 
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            <li style="color: blue;">Consumption analysis of sarcosine, creatine and creatinine by NMR spectroscopy</li>
 
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        </ol>
 
 
     </li>
 
     </li>
 
<li style="color: blue;">Conclusion and Perspectives</li>
 
<li style="color: blue;">Conclusion and Perspectives</li>
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<br>
 
<h2 style="color: blue;"> <b>Usage and Biology</b></h2>
 
<h2 style="color: blue;"> <b>Usage and Biology</b></h2>
  
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<p>KatB is a catalase that “decomposes hydrogen peroxide into water and oxygen; it serves to protect cells from the toxic effects of hydrogen peroxide.” [1] In the context of our project, we wanted to grow <i>Pseudomonas fluorescens</i> in an oxide rich medium so we decided to overexpress this catalase by cloning it into a plasmid and transforming it into the bacteria. We hoped that it would help detoxify the medium as well as detoxify any byproducts caused by other modifications done to the bacteria.</p>
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<p>KatB is a catalase (<a href="https://www.uniprot.org/uniprotkb/C3K2E2/entry" target="blank">EC 1.11.1.6</a>[1]) that decomposes hydrogen peroxide into water and oxygen. In the context of our project, we wanted to grow <i>Pseudomonas fluorescens</i> in an oxide-rich medium. Therefore, we decided to overexpress this catalase to improve stress resistance by cloning its gene into a plasmid for expression in transformed bacteria. We hoped that it would help detoxify the medium as well as detoxify any byproducts caused by other modifications added to the bacteria.</p>
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<br>
 
<h2 style="color: blue;"><b>Sequence and Features</b></h2>
 
<h2 style="color: blue;"><b>Sequence and Features</b></h2>
  
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<p>We decided to amplify the <i>katB</i> gene from the <i>P. fluorescens</i> genome and clone it into the pSEVA244 vector, under control of the <i>Ptrc</i> promoter which is inducible with isopropyl β-D-1-thiogalactopyranoside (IPTG) (<b>Figure 1</b>).</p>
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<p>We amplified the <i>katB</i> gene from the bacterial genome and cloned it into the pSEVA 244 vector, under control of the <i>Ptrc</i> promoter (<a href="https://parts.igem.org/Part:BBa_K3332038" target="blank">BBa_K3332038</a>) which is inducible with IPTG (<b>Figure 1</b>).</p>
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<div align="center">
 
<div align="center">
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             style="width:60%;"
 
             style="width:60%;"
 
             src="https://static.igem.wiki/teams/5108/lea/pseva244-katb.png"><br><br>  
 
             src="https://static.igem.wiki/teams/5108/lea/pseva244-katb.png"><br><br>  
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             <figcaption class="normal"><span class="titre-image"><b>Figure 1: Representation of the pSEVA244-katB plasmid.</b></span></figcaption>
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             <figcaption class="normal"><span class="titre-image"><b>Figure 1: Design of the expression plasmid harboring the <i>katB</i> gene</b></span></figcaption>
 
         </figure>
 
         </figure>
 
</div>
 
</div>
 
<br>
 
<br>
  
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<p>To create the functional vector containing KatB, the cloning of the gene into pSEVA244 linearized was performed following In-Fusion Assembly (Takara). <b>Figure 2</b> demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs R3101S, R3104S). The construct was confirmed by Sanger sequencing (Genewyz, <b>Figure 3</b>).
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<p>To create the expression plasmid containing <i>katB</i>, we cloned the gene into the linearized pSEVA244 backbone by In-Fusion Assembly (Takara, France). <b>Figure 2</b> demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs, France, R3101S, R3104S).
  
 
<div align="center">
 
<div align="center">
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<br>
 
<br>
  
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<div align="center">
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<h2 style="color: blue;"><b>Characterization and Measurements</b></h2>
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        <figure class="normal mx-auto">   
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            <img class="d-block"
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            style="width:100%;"
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            src="https://static.igem.wiki/teams/5108/lea/sanger-sequencing-katb.png"><br><br>
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            <figcaption class="normal"><span class="titre-image"><b>Figure 3: <i>katB</i> locus’ sequencing of pSEVA244-katB plasmid.</b> The <i>katB</i> gene was sequenced by two Sanger sequencing using two flanking primers.</span></figcaption>
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        </figure>
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</div>
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<br>
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 +
<p>The pSEVA438-MBPeGFP plasmid, originally used in <i>P. putida</i> KT2440, was employed as positive control of heterologous protein expression in <i>P. fluorescens</i> SBW25. This construct encodes the fusion protein MBPeGFP (Maltose-Binding Protein enhanced Green Fluorescent Protein) under the control of the <i>Pm</i> promoter inducible by <i>m</i>-toluic acid. Based on the results of <i>Vogeleer P. et al. (2024)</i> [2], the pSEVA438-MBPeGFP- and pSEVA244-katB-transformed <i>P. fluorescens</i> SBW25 strains were cultured in M9 minimal medium supplemented with glucose (28 mM), with or without 0.5 mM of <i>m</i>-toluic acid or IPTG (1 mM). After incubation, a whole-protein extraction was performed for each strain to assess the level of expression, as well as the solubility of our proteins.<br><br>
  
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<h2 style="color: blue;"><b>Characterization and Measurements</b></h2>
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The obtained SDS-PAGE is presented in <b>Figure 3</b>. Both soluble and insoluble fractions contain MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. Although transcriptional leakage was clearly observed without the inducer, MBPeGFP was overproduced when the <i>Pm</i> promoter was activated with 0.5 mM of <i>m</i>-toluic acid, confirming the possibility of heterologous protein expression in <i>P. fluorescens</i> SBW25. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation.<br><br>
  
−
<p>The pSEVA438-MBPeGFP plasmid, originally used in <i>P. putida</i> KT2440, was employed as positive control of heterologous protein expression in <i>P. fluorescens</i> SBW25. This construct encodes the fusion protein MBPeGFP (Maltose-Binding Protein enhanced Green Fluorescent Protein) under the control of the <i>Pm</i> promoter. Based on the results of <i>Vogeleer P. et al. (2024)</i> [2], the pSEVA438-MBPeGFP- and pSEVA244-katB-transformed <i>P. fluorescens</i> SBW25 strains were cultured in M9 minimal medium supplemented with glucose (28 mM), with or without 0.5 mM of <i>m</i>-toluic acid or IPTG (1 mM). After incubation, a whole-protein extraction was performed for each strain to assess the level of expression, as well as the solubility of our proteins.</p>  
+
SDS-PAGE analysis of the cell lysate derived from <i>P. fluorescens</i> transformed with pSEVA244 revealed a visible band at the expected size of KatB only in insoluble fractions when its expression is induced.</p>
  
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<p>The obtained SDS-PAGE is presented in <b>Figure 4</b>. Both soluble and insoluble fractions contain MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. Although transcriptional leakage was clearly observed without the inducer, MBPeGFP was overproduced when the <i>Pm</i> promoter was activated with 0.5 mM of <i>m</i>-toluic acid, confirming the possibility of heterologous protein expression in <i>P. fluorescens</i>. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation.<br>
 
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COMPLETER AVEC RESULTATS KATB</p>
 
  
 
<div align="center">
 
<div align="center">
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             style="width:60%;"
 
             style="width:60%;"
 
             src="https://static.igem.wiki/teams/5108/lea/sds-page-katb.png"><br><br>  
 
             src="https://static.igem.wiki/teams/5108/lea/sds-page-katb.png"><br><br>  
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             <figcaption class="normal"><span class="titre-image"><b>Figure 4: SDS-PAGE of soluble and insoluble protein fractions from cultures of <i>Pseudomonas fluorescens</i> transformed with pSEVA438-MBPeGFP or pSEVA244-katB.</b> <i>P. fluorescens</i> was cultured with and without inducer, <i>m</i>-toluic acid. Arrows show expected size of MBPeGFP and the catalase (KatB).</span></figcaption>
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             <figcaption class="normal"><span class="titre-image"><b>Figure 3: SDS-PAGE of soluble and insoluble protein fractions from cultures of <i>Pseudomonas fluorescens</i> transformed with pSEVA438-MBPeGFP or pSEVA244-katB.</b> <i>P. fluorescens</i> was cultured with and without inducer, <i>m</i>-toluic acid. Arrows indicate the expected size of catalase KatB.</span></figcaption>
 
         </figure>
 
         </figure>
 
</div>
 
</div>
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<h2 style="color: blue;"><b>Conclusion and Perspectives</b></h2>
 
<h2 style="color: blue;"><b>Conclusion and Perspectives</b></h2>
  
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<p>After looking at the 3D representation of this protein, we realized that it has a peptide signal, meaning that the protein is being secreted outside of the cell. If the protein is being excreted outside of the cell, it could aid plants survive on regolith by deoxidizing the medium. In order to prove that the engineered bacterium can better survive certain conditions, the oxidative impact test should be performed again as well as a viability assay on regolith. Plant growth should also be tested in the presence of <i>P.fluorescens</i> pSEVA244-KatB. Hopefully, plants will live longer and in better conditions with the overexpression of the catalase.</p>
+
<p>The presence of proteins in the insoluble fraction suggests that the ​​conditions of production (e.g., inducer concentration and temperature) are not optimal for overexpressing KatB in the soluble form. After aligning the sequence of KatB with database PFAM, we noticed the presence of a peptide signal, suggesting the targeting of KatB in the periplasm. In our lysis protocol, we could not differentiate periplasmic proteins from cytoplasmic ones. To solve this problem, we could change the KatB production conditions and our lysis protocol to purify exclusively periplasmic proteins.</p>
  
  
 +
<br>
 
<h2 style="color: blue;"><b>References</b></h2>
 
<h2 style="color: blue;"><b>References</b></h2>
  
 
<ol>
 
<ol>
 
     <i>
 
     <i>
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<li>Washington IM & Van Hoosier G (2012) Chapter 3 - Clinical Biochemistry and Hematology. In The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents, Suckow MA Stevens KA & Wilson RP (eds) pp 57–116. Boston: Academic Press
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<li>UniProt. (s. d.-d). https://www.uniprot.org/uniprotkb/C3K2E2/entry</i>
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    <li>Tsuru D, Oka I & Yoshimoto T (1976) Creatinine Decomposing Enzymes in Pseudomonas putida. Agricultural and Biological Chemistry 40: 1011–1018 </li>
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<li>Vogeleer P, Millard P, Arbulú A-SO, Pflüger-Grau K, Kremling A & Létisse F (2024) Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control. Metabolic Engineering 81: 26–37</li>
−
    <li>Vogeleer P, Millard P, Arbulú A-SO, Pflüger-Grau K, Kremling A & Létisse F (2024) Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control. Metabolic Engineering 81: 26–37</li>
+
 
</i>
 
</i>
 
</ol>
 
</ol>

Latest revision as of 16:03, 29 September 2024


Catalase from Pseudomonas fluorescens SBW25

Catalse from P. fluorescens SBW25


    Contents
  1. Usage and Biology
  2. Sequence and Features
  3. Characterization and Measurements
  4. Conclusion and Perspectives
  5. References



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 917
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 148
    Illegal NgoMIV site found at 322
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 723
    Illegal SapI.rc site found at 1240


Usage and Biology

KatB is a catalase (EC 1.11.1.6[1]) that decomposes hydrogen peroxide into water and oxygen. In the context of our project, we wanted to grow Pseudomonas fluorescens in an oxide-rich medium. Therefore, we decided to overexpress this catalase to improve stress resistance by cloning its gene into a plasmid for expression in transformed bacteria. We hoped that it would help detoxify the medium as well as detoxify any byproducts caused by other modifications added to the bacteria.


Sequence and Features

We amplified the katB gene from the bacterial genome and cloned it into the pSEVA 244 vector, under control of the Ptrc promoter (BBa_K3332038) which is inducible with IPTG (Figure 1).



Figure 1: Design of the expression plasmid harboring the katB gene

To create the expression plasmid containing katB, we cloned the gene into the linearized pSEVA244 backbone by In-Fusion Assembly (Takara, France). Figure 2 demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs, France, R3101S, R3104S).



Figure 2: Restriction digest of pSEVA244-katB plasmid. The plasmid was digested with EcoRI and HindIII separately or in combination. The expected (left) and experimental (right) digestion patterns are shown.

Characterization and Measurements

The pSEVA438-MBPeGFP plasmid, originally used in P. putida KT2440, was employed as positive control of heterologous protein expression in P. fluorescens SBW25. This construct encodes the fusion protein MBPeGFP (Maltose-Binding Protein enhanced Green Fluorescent Protein) under the control of the Pm promoter inducible by m-toluic acid. Based on the results of Vogeleer P. et al. (2024) [2], the pSEVA438-MBPeGFP- and pSEVA244-katB-transformed P. fluorescens SBW25 strains were cultured in M9 minimal medium supplemented with glucose (28 mM), with or without 0.5 mM of m-toluic acid or IPTG (1 mM). After incubation, a whole-protein extraction was performed for each strain to assess the level of expression, as well as the solubility of our proteins.

The obtained SDS-PAGE is presented in Figure 3. Both soluble and insoluble fractions contain MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. Although transcriptional leakage was clearly observed without the inducer, MBPeGFP was overproduced when the Pm promoter was activated with 0.5 mM of m-toluic acid, confirming the possibility of heterologous protein expression in P. fluorescens SBW25. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation.

SDS-PAGE analysis of the cell lysate derived from P. fluorescens transformed with pSEVA244 revealed a visible band at the expected size of KatB only in insoluble fractions when its expression is induced.



Figure 3: SDS-PAGE of soluble and insoluble protein fractions from cultures of Pseudomonas fluorescens transformed with pSEVA438-MBPeGFP or pSEVA244-katB. P. fluorescens was cultured with and without inducer, m-toluic acid. Arrows indicate the expected size of catalase KatB.

Conclusion and Perspectives

The presence of proteins in the insoluble fraction suggests that the ​​conditions of production (e.g., inducer concentration and temperature) are not optimal for overexpressing KatB in the soluble form. After aligning the sequence of KatB with database PFAM, we noticed the presence of a peptide signal, suggesting the targeting of KatB in the periplasm. In our lysis protocol, we could not differentiate periplasmic proteins from cytoplasmic ones. To solve this problem, we could change the KatB production conditions and our lysis protocol to purify exclusively periplasmic proteins.


References

  1. UniProt. (s. d.-d). https://www.uniprot.org/uniprotkb/C3K2E2/entry
  2. Vogeleer P, Millard P, Arbulú A-SO, Pflüger-Grau K, Kremling A & Létisse F (2024) Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control. Metabolic Engineering 81: 26–37