Difference between revisions of "Part:BBa K3589105"
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This basic part contains the coding sequence of the mutant form L499F of the laccase from the ascomycete <i>Botrytis aclada</i> (hereafter referred to as BaLac). This part is codon-optimized for <i>Escherichia coli</i>. Combined a promoter and a terminator, this basic part mediates the oxidation of a wide variety of substrates including phenolic compounds and aromatic amines. This part can be cloned into standard <I>E coli</I>. expression vectors like the commercially available pGEX-6P-1 vector.  
 
This basic part contains the coding sequence of the mutant form L499F of the laccase from the ascomycete <i>Botrytis aclada</i> (hereafter referred to as BaLac). This part is codon-optimized for <i>Escherichia coli</i>. Combined a promoter and a terminator, this basic part mediates the oxidation of a wide variety of substrates including phenolic compounds and aromatic amines. This part can be cloned into standard <I>E coli</I>. expression vectors like the commercially available pGEX-6P-1 vector.  
 
The mutant laccase L499F has a high redox potential and shows activity near neutral pH (Scheiblbrandner et. al, 2017; doi: 10.1038/s41598-017-13734-0).
 
The mutant laccase L499F has a high redox potential and shows activity near neutral pH (Scheiblbrandner et. al, 2017; doi: 10.1038/s41598-017-13734-0).
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<b>Summary of the Results from Team Kaiserslautern 2019</b><br>
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<li>Expression could be achieved in different <i>E. coli</i> strains</li>
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<li>Protein showed activity in ABTS-assay</li>
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<li>Protein could degrade Diclofenac</li>
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<u><b>Design of the constructs</b></u><br><br>
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<p>For the recombinant expression of the laccase genes from <i>Botritis aclada</i>(<i>baLac</i>) the <i>E. coli</i> vector pGEX-6P-1 was used (<b>Fig. 1</b>). This expression vector is used to construct a translation fusion protein of Glutathione S-transferase (GST) and our two different laccases. The expression is regulated by a tac promotor. This promotor combines the strong expression rate from the tryptophan promotor and can be induced with IPTG like the lac operon. To make sure the promotor is inhibited if there is no induction with IPTG the vector also includes the genetic code for the lac-inhibitor that can bind the lac-operon. Because GST has a high affinity for glutathione, the fusion of the laccases with GST allows the purification by affinity chromatography using glutathione agarose. In addition, a protease cleavage site is incorporated between the GST and our fusion protein (BaLac-GST). This allows the separation of GST and the laccase using PreScission Protease. BaLac has a size of 89.3 kDa with GST and a size of 61.6 kDa without GST. For selection of plasmid containing cells the expression vector carries an ampicillin resistance gene.<br>
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In order to be able to do further enzyme assays with the laccases, the vector pGEX-6P-1-<i>balac</i> has to be transformed in the <i>E. coli</i> expression strain BL21(DE3). Additionally, we transformed them in <i>E. coli</i> DH5α for isolation the vectors. The strain has an <i>endA1</i> mutation. This leads to the inactivity of an intracellular endonuclease, which degrades plasmid DNA. Therefore, plasmid DNA isolation is more efficient.</p><br>
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<img src="https://2020.igem.org/wiki/images/5/54/T--TU_Kaiserslautern--VEC_BA1.png>
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<br><p><b>Fig. 1: pGEX-6P-1_<i>baLac </i>.</b> It includes a <i>tac<i> promotor that can be regulated with the lac-inhibitor (expressed with lacI) which can be inactivated with IPTG. The protein is fused to GST, with a PreScission protease cleavage site in between. The vector has an ampicillin resistance gene, which is used for selection. </p><br><br>
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Revision as of 13:33, 27 October 2020


Mutant laccase from Botrytis aclada for Escherichia coli

This basic part contains the coding sequence of the mutant form L499F of the laccase from the ascomycete Botrytis aclada (hereafter referred to as BaLac). This part is codon-optimized for Escherichia coli. Combined a promoter and a terminator, this basic part mediates the oxidation of a wide variety of substrates including phenolic compounds and aromatic amines. This part can be cloned into standard E coli. expression vectors like the commercially available pGEX-6P-1 vector. The mutant laccase L499F has a high redox potential and shows activity near neutral pH (Scheiblbrandner et. al, 2017; doi: 10.1038/s41598-017-13734-0).



Summary of the Results from Team Kaiserslautern 2019

  • Expression could be achieved in different E. coli strains
  • Protein showed activity in ABTS-assay
  • Protein could degrade Diclofenac

Design of the constructs

For the recombinant expression of the laccase genes from Botritis aclada(baLac) the E. coli vector pGEX-6P-1 was used (Fig. 1). This expression vector is used to construct a translation fusion protein of Glutathione S-transferase (GST) and our two different laccases. The expression is regulated by a tac promotor. This promotor combines the strong expression rate from the tryptophan promotor and can be induced with IPTG like the lac operon. To make sure the promotor is inhibited if there is no induction with IPTG the vector also includes the genetic code for the lac-inhibitor that can bind the lac-operon. Because GST has a high affinity for glutathione, the fusion of the laccases with GST allows the purification by affinity chromatography using glutathione agarose. In addition, a protease cleavage site is incorporated between the GST and our fusion protein (BaLac-GST). This allows the separation of GST and the laccase using PreScission Protease. BaLac has a size of 89.3 kDa with GST and a size of 61.6 kDa without GST. For selection of plasmid containing cells the expression vector carries an ampicillin resistance gene.
In order to be able to do further enzyme assays with the laccases, the vector pGEX-6P-1-balac has to be transformed in the E. coli expression strain BL21(DE3). Additionally, we transformed them in E. coli DH5α for isolation the vectors. The strain has an endA1 mutation. This leads to the inactivity of an intracellular endonuclease, which degrades plasmid DNA. Therefore, plasmid DNA isolation is more efficient.