Difference between revisions of "Part:BBa K2043007"
Line 2: Line 2:
 
<partinfo>BBa_K2043007 short</partinfo>
 
<partinfo>BBa_K2043007 short</partinfo>
  
−
This part corresponds to <b><i>Bacillus pumilus</i> laccase</b> cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project(<a href="https://parts.igem.org/Part:BBa_K2043001:Design"/>). This enzymes originally comes from <i>Bacillus pumilus</i>, which we <b>codon optimised for <i>E. coli</i></b>.<br>
+
This part corresponds to <b><i>Bacillus pumilus</i> laccase</b> cloned by the Paris Bettencourt team in 2016 in the context of the (<a href="https://parts.igem.org/Part:BBa_K2043001:Design">Frank&Stain project</a>). This enzymes originally comes from <i>Bacillus pumilus</i>, which we <b>codon optimised for <i>E. coli</i></b>.<br>
 
In order to facilitate working with this enzyme, we added a <b>His-tag</b> at the <b>C-terminal</b>. This tag allows for purification in an easier way.<br><br>
 
In order to facilitate working with this enzyme, we added a <b>His-tag</b> at the <b>C-terminal</b>. This tag allows for purification in an easier way.<br><br>
  
−
This gene had already been registered in the Biobrick Registry, in part BBa_K863000.<br><br>
+
This gene has already been registered in the Biobrick Registry (part number:BBa_K863000.)<br><br>
 +
 
 +
There are several reasons why we have chosen this enzyme:
 +
<ol>
 +
<li>It already exists in the registry of Standard Biological Parts, and it is one of the most well documented BioBricks.</li>
 +
<li>Apart from the registry, a good biochemical analysis and methods can be found in the paper by Reiss et. al 2011.</li>
 +
<li>The laccasse comes from bacteria (Bacillus pumilus) which makes it potentially easier to express and study in E. coli than fungal lacasses.</li>
 +
<li>To our knowledge, bpul hasn't been shown to degrade indigo. On the other hand Reiss et. Al 2011. have shown that it successfully degrades indigo carmine which is structurally similar to indigo dye (figure 4A).</li>
 +
<li>Due to a large number of substrates laccases can potentially degrade compounds from wine making it a good basis for collaboration with the Enzyme Group.</li>
 +
</ol>
  
−
We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. Our project consisted in the degradation of wine strains, and therefore enzymes with the ability to degrade polyphenolic molecules were of interest to us. <br>
 
−
In particular, laccases are good candidates because they are known to degrade Anthocyanins.<br><br>
 
  
 
<b>Testing the part</b><br><br>
 
<b>Testing the part</b><br><br>

Revision as of 15:17, 25 October 2016

bpuI laccase codon optimized for E. coli

This part corresponds to Bacillus pumilus laccase cloned by the Paris Bettencourt team in 2016 in the context of the (<a href="https://parts.igem.org/Part:BBa_K2043001:Design">Frank&Stain project</a>). This enzymes originally comes from Bacillus pumilus, which we codon optimised for E. coli.
In order to facilitate working with this enzyme, we added a His-tag at the C-terminal. This tag allows for purification in an easier way.

This gene has already been registered in the Biobrick Registry (part number:BBa_K863000.)

There are several reasons why we have chosen this enzyme:

  1. It already exists in the registry of Standard Biological Parts, and it is one of the most well documented BioBricks.
  2. Apart from the registry, a good biochemical analysis and methods can be found in the paper by Reiss et. al 2011.
  3. The laccasse comes from bacteria (Bacillus pumilus) which makes it potentially easier to express and study in E. coli than fungal lacasses.
  4. To our knowledge, bpul hasn't been shown to degrade indigo. On the other hand Reiss et. Al 2011. have shown that it successfully degrades indigo carmine which is structurally similar to indigo dye (figure 4A).
  5. Due to a large number of substrates laccases can potentially degrade compounds from wine making it a good basis for collaboration with the Enzyme Group.


Testing the part

We tested the activity of BpuI using cell extract of cells expressing our protein.
First, we performed an SDS-PAGE to check whether the protein was being expressed.

Paris_Bettencourt_notebook_GELS.jpg

The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity.
We tested our cell extract for BpuI activity in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS being used as substrate, as recommended in the literature.
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product.

Paris_Bettencourt_notebook_bpuI_good.jpg

As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product at 420nm, which results from the oxidation of ABTS. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 888
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 888
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 888
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 888
    Illegal AgeI site found at 52
    Illegal AgeI site found at 247
  • 1000
    COMPATIBLE WITH RFC[1000]


Cho, E. A., Seo, J., Lee, D. W., & Pan, J. G. (2011). Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores. Enzyme and microbial technology, 49(1), 100-104.

Reiss, R., Ihssen, J., & Thöny-Meyer, L. (2011). Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC biotechnology, 11(1), 1.