Difference between revisions of "Part:BBa K2043004"

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−	This part corresponds to the gene coding for the protein Catechol-2,3-dioxygenase (<i>xylE</i>) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzyme catalyses the following chemical reaction with EC number 1.13.11.2	+	This part corresponds to the gene coding for the protein Catechol-2,3-dioxygenase (<i>xylE</i>) cloned by the Paris Bettencourt team in 2016 in the context of the <a href="http://2016.igem.org/Team:Paris_Bettencourt">Frank&Stain project</a>. This enzyme catalyses the following chemical reaction with EC number 1.13.11.2
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Revision as of 15:50, 27 October 2016

xylE from Pseudomonas putida codon optimized for E. coli

Description

This part corresponds to the gene coding for the protein Catechol-2,3-dioxygenase (xylE) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzyme catalyses the following chemical reaction with EC number 1.13.11.2


Figure 1 oxidation of catechol to 2-hydroxymuconate semialdehyde taken from wikipedia commons, created by user Yikrazuul.

The figure 1 shows of catechol 2,3-dioxygenases catalyzes the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds are structurally similar to catechol, making Catechol-2,3-dioxygenase a good candidate for anthocyanin degradation. Catechol-2,3-dioxygenase is also found in many species of soil bacteria. This enzymes originally comes from Pseudomonas putida (NCBI Ref. Seq.: NP_542866.1), which we codon optimized for E. Coli and avoided the BsaI restriction sites. An His-tag was also added at the C-terminal. This tag allows for purification in an easier way.
We wanted to test Catechol-dioxygenases: one was XylE from Pseudomonas putida , which uses catechol as a main substrate. We hypothesized that this enzyme would be a strong candidate for removal of red-wine stains because catechol shares important structural similarities with anthocyanin (Cerdan 1995, Kobayasi 1995 and Lin 2015).

Testing the part

We tested the expression and activity of XylE using cell extract from E.coli transformants overexpressing our protein. First, we performed an SDS-PAGE to check whether the protein was successfully expressed.
Figure 2 SDS-PAGE gels for expression of BpuI, CatA and XylE. Sample preparation: E.coli strain BL21(DE3), expressing the proteins, were induced for 5 hours with 0.5mM IPTG. After 5 hours, the OD(600nm) was measured, the cells centrifuged and the pellet was resuspended in Laemmli sample buffer (from BIO-RAD) to a final OD600 of 10. The cells were cooked at 95ºC for 10 min, and 10uL of the resulting solution were loaded on the gel. Ladder used: Kaleidoscope. Staining: The gel was washed 3x with miliQ water to remove the SDS and staining was performed using BioRad Comassie for 30min. De-staining was performed by leaving the gel in miliQ water for 1 hour with gentle shaking. As expected, no overexpression was observed in BL21(DE3), whether it was induced or not induced. BpuI was correctly overexpressed, with the observed size being the expected, around 59kDa. CatA overexpression was very mild, but the correct sized band was observed, of around 34kDa. XylE was correctly overexpressed, with the correct band being observed, of around 36kDa.

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 XylE activity in Sodium Phosphate 50mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Control corresponds to cells which do not express our proteins. In all cases, values measured correspond to reaction product.


Figure 3 Absorbance of the reaction product, 2-hydroxymunonic semialdehyde. The absorbance of the product was measured at 375nm over a period of time in order to follow the activity of the reaction of XylE. The blue line represents the negative control, and the green line shows the activity of the cell extract containing XylE.


Figure 4 This barplot is the result of the analysis of the data presented in figure 3. XylE activity was measured in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed.

As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product, (2-hydroxymunonic semialdehyde), at 375nm. Since much more reaction product is produced with cells expressing XylE than in the control, we can affirm that the enzyme was functional.

References

Kobayashi, T., Ishida, T., Horiike, K., Takahara, Y., Numao, N., Nakazawa, A., ... & Nozaki, M. (1995). Overexpression of Pseudomonas putida catechol 2, 3-dioxygenase with high specific activity by genetically engineered Escherichia coli. Journal of biochemistry, 117(3), 614-622.

Cerdan, P., Rekik, M., & Harayama, S. (1995). Substrate Specificity Differences Between Two Catechol 2, 3‐Dioxygenases Encoded by the TOL and NAH Plasmids from Pseudomonas putida. European journal of biochemistry, 229(1), 113-118.

Sequence and Features