Part:BBa_K2043007

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.) All the results presented here for bpul have been tested in pCOLA vector.

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.

Over-expression of bpul

SDS-PAGE was performed to check whether the protein was successfuly expressed.

<img src="" width="500px"> Figure 1 Over-expression of bpul. The BL21(DE3)-lacZ is used as a negative control. This strain expresses alpha subunit of lacZ gene instead of Bpul. The left side of the figure is showing the results of the electrophoresis of the cell extracts at the moment of adding IPTG. The right side are the results 5h after the addition of IPTG. We can see the successful expression of bpul and the leakiness of the promoter.

<img src="" width="500px"> Figure 2 The over-expression of bpul alongside the other two enzymes used in Frank&Stain project.

Testing the 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.

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.

We tested the activity of BpuI using cell extract of cells expressing our protein.