Team Aboa 2021


New documentation to an existing Part

Since we used the CotA sequence that the Team TU Darmstadt 2020 had uploaded to the Part Registry, we decided to add the results that we obtained with CotA to that Part page (BBa_K3429011). In addition, we added new information from literature to the Main Page of another already existing Part. That Part was “Blue copper oxidase CueO” (BBa_K3429012) that the Team TU Darmstadt 2020 had originally uploaded to the Part Registry. This is the information we added:


Laccases are multi-copper oxidases (MCOs) that can catalyze reactions of many substrates, for example organic compounds, dyes and pharmaceuticals. They can be found in fungi, bacteria, insects and plants and they are involved in many different functions, for example in lignin degradation, pigmentation, pathogenesis of fungi and wound healing in plants. [2] CueO is a multicopper oxidase laccase produced by E. coli [3].

In vitro, CueO can oxidise various compounds, such as catechols, ferrous iron and iron-chelating siderophores [4]. It has been proven that CueO plays a pivotal role in regulating the copper resistance of E. coli [5]. The mechanism of action of CueO in vivo has been suggested to be the cuprous oxidation in which CueO confers more toxic Cu(I) to less toxic Cu(II). According to one study, the optimal pH for the proper CueO function is 6,5. [3]

Coppers play a major role in the oxidation reactions of MCOs due to their central location in catalytic sites. It has also been previously shown that the oxidation activity of CueO depends on copper concentrations. [6] In general, MCOs contain four copper atoms in different sites; in a T1 site (“blue” copper), in a T2 site (“normal” copper) and in two types of T3 sites (“binuclear” coppers) [3]. When it comes to a reaction of CueO, the T1 site acts as an electron transfer site whereas T2 and T3 perform dioxygen reduction [7]. Unlike other multicopper oxidases, CueO requires five copper atoms and it has a methionine-rich helix that functions as a valuable regulative element regarding copper binding [4]. In the absence of copper, that helix acts by blocking access to the T1 site and simultaneously providing a new copper-binding site T4 [3,7]. It has been shown that the deletion of this helix leads to a remarkably decreased oxidation activity which highlights the importance of the T4 site in the CueO function. In Figure 1, you can see the proposed reaction mechanism for CueO oxidative function illustrated with an example substrate compound [Cu(I)(Bca)2]3- in BisTris Buffer. [7]

A figure of a four-phase  mechanism of CueO as a cuprous oxidative agent.
Figure 1. The proposed mechanism of CueO as a cuprous oxidative agent. In this example, the substrate is [Cu(I)(Bca)2]3- in the BisTris Buffer. The red sphere indicates a Cu(I) atom whereas the blue sphere indicates a Cu(II) atom. The picture illustrates how the T4 site changes from an empty resting state (i) to the copper-binding (ii-iii) and copper-oxidating states (iv). [7]


The structure of CueO has been succeeded to determine at 1,4 Å resolution, which allows a precise positioning of copper binding sites. CueO is composed of three pseudoazurin domains. Both the T1 site and a methionine-rich helix are located in domain 3. [3] The methionine-rich helix provides an extra copper-binding site T4, which is only 7.5 Å away from the T1 site [7].


  • [2] L. Arregui et al. Laccases: structure, function, and potential application in water bioremediation. Microbial Cell Factories 2009, 18 (1) 200, doi: 10.1186/s12934-019-1248-0.
  • [3] S. Roberts et al. Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 2002, 99 (5) 2766-2771, doi: 10.1073/pnas.052710499.
  • [4] S. Singh et al. Cuprous Oxidase Activity of CueO from Escherichia coli. Journal of Bacteriology 2004, 186 (22) 7815-7817, doi: 10.1128/JB.186.22.7815-7817.2004.
  • [5] G. Grass and C. Rensing. CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli. Biochemical and biophysical research communications 2001, 286 (5) 902-908, doi: 10.1006/bbrc.2001.5474.
  • [6] X. Li et al. Crystal structures of E. coli laccase CueO at different copper concentrations. Biochemical and biophysical research communications 2007, 354 (1) 21-26, doi: 10.1016/j.bbrc.2006.12.116.
  • [7] K. Djoko et al. Reaction Mechanisms of the multicopper oxidase CueO from Escherichia coli support its functional role as a cuprous oxidase. Journal of the American Chemical Society 2010, 132 (6) 2005-2015, doi: 10.1021/ja9091903.”

New Parts to the Registry

In addition to adding new information to an existing Part, we also added two Parts to the Registry. The Parts we added were following:

  • Multi-copper oxidase Yak from Yersinia enterocolitica (BBa_K3872000)
  • Multi-copper oxidase CueO from Escherichia coli (BBa_K3872001)

We also added our results to the Experiment Page of these Parts. The reason we added our own CueO laccase to the Registry although there was already one CueO Part by Team Darmstadt 2020 was that our laccase had minor changes when compared to the existing one.

Guidebook for future teams

We created a How to form a team and start a project -guidebook for future iGEM teams to help them avoid all the problems we have had. In the guidebook we share our story and provide hints to the following things:

  • Forming a new team: What kind of skills are required and what are advisors and PIs?
  • Starting fundraising: When to start and where to ask for money?
  • Starting brainstorming: What are the most important things and what kind of exercises may help you?

In this guidebook you can find all the crucial steps to start a successful iGEM project.

Phototroph Handbook Chapter

We also contributed to the Phototroph Handbook by writing a chapter in collaboration with team Toulouse_INSA-UPS. The chapter was about transformation methods with cyanobacteria and it can be read in the pdf provided below. Our chapter is an important contribution to future iGEM teams as it facilitates the utilization of cyanobacteria as a project chassis. Together with the rest of the handbook we hope that it inspires future teams to choose to work with phototrophic organisms.