Difference between revisions of "Part:BBa K5108002"
Line 47: Line 47:
 
<br>
 
<br>
  
−
<p>To create the functional vector containing <i>katB</i>, the cloning of the gene into pSEVA244 linearized was performed following In-Fusion Assembly (Takara, France). <b>Figure 2</b> demonstrates the successful cloning by restriction digest with EcoRI and HindIII enzymes (New England Biolabs R3101S, France, R3101S, R3104S). The construct was confirmed by Sanger sequencing (Genewyz, Germany, <b>Figure 3</b>).
+
<p>To create the functional vector containing <i>katB</i>, the cloning of the gene into pSEVA244 linearized was performed following 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). The construct was confirmed by Sanger sequencing (Genewyz, Germany, <b>Figure 3</b>).
  
 
<div align="center">
 
<div align="center">
Line 72: Line 72:
 
<h2 style="color: blue;"><b>Characterization and Measurements</b></h2>
 
<h2 style="color: blue;"><b>Characterization and Measurements</b></h2>
  
−
<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> [1], 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>
+
<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> [1], 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>
  
−
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>
+
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>
  
 
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>
 
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>

Revision as of 07:37, 28 September 2024


Catalase from Pseudomonas fluorescens SBW25

P. fluorescens catalase KatB ORF


    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) that "decomposes hydrogen peroxide into water and oxygen. It serves to protect cells from the toxic effects of hydrogen peroxide." In the context of our project, we wanted to grow Pseudomonas fluorescens in an oxide rich medium so we decided to overexpress this catalase to improve stress resistance by cloning its native gene 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 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 isopropyl β-D-1-thiogalactopyranoside (IPTG) (Figure 1).



Figure 1: Representation of the pSEVA244-katB plasmid.

To create the functional vector containing katB, the cloning of the gene into pSEVA244 linearized was performed following 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). The construct was confirmed by Sanger sequencing (Genewyz, Germany, Figure 3).



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.



Figure 3: katB locus’ sequencing of pSEVA244-katB plasmid. The katB gene was sequenced by two Sanger sequencing using two flanking primers.

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) [1], 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 4: 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 show expected size of MBPeGFP and the catalase (KatB).

Conclusion and Perspectives

Presence in insoluble fraction can suggest that the ​​conditions of production (inducer concentration / temperature etc…) are not optimal to overexpress KatB in soluble form. After aligning the sequence of KatB with PFAM database, we observe the presence of a peptide signal. Research shows that enzymes with similar structures and peptide signals are periplasmic. We concluded that our ⁠protein could be located in the periplasm which means that it could have been recovered in the insoluble fraction, even if it was active. Overall, in our lysis protocol, we could not differentiate periplasmic proteins from cytoplasmic ones. We could not conclude on whether we had successfully overproduced KatB. To solve these problems, we could have changed katB production conditions and our lysis protocol to be able to see only periplasmic proteins.

References

  1. 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
    2.