Coding

Part:BBa_K2043007

Designed by: Mislav Acman   Group: iGEM16_Paris_Bettencourt   (2016-10-13)

bpuI laccase codon optimized for E. coli 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal PstI site found at 888

This part corresponds to Bacillus pumilus laccase cloned by the Paris Bettencourt team in 2016 in the context of the (Frank&Stain project). The gene originates 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, allowing for protein purification.
Laccases are commonly used enzymes for bleaching denim, as well as other textiles. They act in part by oxidizing phenolic compounds, offering a similar target to the phenol ring disrupting catalase enzymes also developed in this project (BBa_K2043001) (Reiss, 2011).
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 laccase comes from bacteria (Bacillus pumilus) which makes it potentially easier to express and study in E. coli than fungal laccases.
  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. The large number of substrates degraded by laccases make this enyzme a good candidate for degrading anthocyanins.

Over-expression of bpul

SDS-PAGE electrophoresis was performed on cell extracts to confirm the protein expression.

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.

Figure 2 The over-expression of bpul alongside the other two enzymes used in Frank&Stain project.

Testing the Activity

ABTS degradation

We tested our cell extracts containing Bpul laccase for ability to oxidize ABTS which is usually used to test activities of laccases. The experiments were performed in in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS.


Figure 3 Plot on the left shows the absorbance measurements of ABTS oxidation at wavelength 420 nm. The negative control (BL21(DE3)) is showing no activity. However, bpul demonstrates high activity which reaches saturation after less than 2 h. In the right figure there is a clear difference between activity of our enzyme and the control.

Indigo and indigo carmine degradation

The transformant BL21(DE3)-bpul, and the negative control BL21(DE3) have been induced with IPTG. The cell extracts of both strains were tested for the ability to degrade indigo carmine (figure 4 right) and indigo (figure 4 left). Additionally, 10µM acetosyringone (ACS) was added to the reaction mixture to facilitate the oxidation of the substrates. ACS is a known mediator of laccases and it is often used in the studies of laccase activitiy. As expected from experience of the previous experiments (Reiss 2011), the 1mM indigo carmine solution is entirely degraded within the 13 hours of the experiment in the cell extract which contains overexpressed bpul. Additionally, we have shown that more than 60% of indigo can be degraded by bpul laccase in the 13 hours of the experiment. As our degradation curve did not reach saturation, it is probable that longer incubation would lead to higher levels of degradation.

Figure 4 Indigo carmine and indigo degradation by cell extract containing Bpul are shown over a period of 13h. The experiment was carried out in 96-well plate at λ=650 for indigo carmine, and λ=680 nm for indigo, at room temperature. 1 µL of cell extract was added to the reaction mixture which consisted of substrate dissolved in 0.1 mM of potassium phosphate buffer (pH 7.8), and 10 µM ACS added to the reaction mix.

References

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.

[edit]
Categories
Parameters
None