Team:EPFL/Engineering Success

• Project
  • Description
    Project background, inspiration, etc.
  • Background
    Quantification of the problem
  • Design
    Monomer and dimer design
  • Modelling
    Bioreactor fluid mechanics models
  • Results
    Main results of the project
  • Engineering Success
    Design, build, test, learn
  • Implementation
    Bioreactor
  • Proof of Concept
    Showing yeast captures copper
  • Contribution
    Contribution to future teams
  • Safety
    Chemicals used, security measures
  • Protocols
    PCR, Western Blot, transformation, etc.
  • Notebook
    Detailed records of all experiments
• People
  • Team
  • Attributions
  • Collaborations
• Parts
  • Parts Overview
  • Parts Collection
• Human Practices
  • Human Practices
  • Integrated Human Practices
  • Education & Communication
• Medals

Engineering Success

Contents

• Introduction
• Design
• Build
• Test
• Learn
• Design

Introduction

Engineering a synthetic biological system is not a straightforward but rather complex path made with many moves back and forth and intricate thought processes. We believe this process is best represented as an engineering cycle. The typical engineering cycle consists of 4 steps that are repeated indefinitely : the design, build, test and learn phases. As students in an engineering school we are inclined to tackle our synthetic biology problem with an engineering approach. On this page, we attempt to demonstrate our engineering cycle throughout our project.

Design

Once we found our subject and problem to solve, that being copper contamination in agricultural fields, there came the time to design a solution. After brainstorming the different options we could identify as potential candidates in pre-existing academic literature, we started designing our project: yeast displaying copper metallothionein proteins at its membrane surface through a commonly used display system. This idea came from the fact that yeast have endogenous bio-accumulation abilities, in particular through the action of their endogenous metal binding proteins, or metallothioneins, capable of keeping heavy metals from causing harm to the rest of the organism by trapping them in a pocket. We thought of improving this natural function by expressing the copper metallothionein protein CUP1 extracellularly, thus allowing the yeast to bind copper ions on its external surface. In addition to improving its capacity to fix copper, this design would override the accumulation limit that yeast encountered due to the toxicity of copper. This was version 1 of the project. In order to bind even more copper ions, we thought of expressing a multimer of CUP1, each protein being fused together by a sequence of amino acids called a linker. We started with dimers (two copies of CUP1) and this was version 2 of the project. A more detailed explanation of the design process and choice of the different linkers can be found on the Design page.

Build

After designing the project we could move on to the next step which was bringing our ideas to life. We cloned the CUP1 endogenous gene in a plasmid allowing for extracellular membrane expression in yeast as well as our designed CUP1 dimers and cultured our chassis : the EBY100 yeast strain. We transformed the microorganism with the recombinant plasmid and could move on to the testing phase. All the steps in the “building” process were individually tested in order to control the process of cloning and transformation. These are detailed in the laboratory notebooks.

Test

Once designed and built, a system needs to be tested. To do so, we have specifically designed an experiment to measure copper concentrations when copper is put in solution with yeast and thus, measure the absorption of copper ions by the yeast. We grew yeast until a defined concentration of cells and diluted it in a known concentration of copper solution. We then collected samples at multiple time points, filtered the yeast by centrifugation, and then measured the copper concentration in the supernatant. We tested multiple variables in the experiment by changing either the yeast concentration, the initial copper concentration and compared different yeast strains. The details and results of this testing process can be found here.

Learn

Unfortunately, the results obtained during our testing phase were not what we expected. Indeed, our engineered yeast did not improve the endogenous bio-accumulation function of the wild type yeast. A more detailed picture of the results is depicted on the Results page, where you can also find a discussion of what could explain this unexpected result. In the end, we succeeded in making the engineered yeast work almost as well as the wild type in absorbing copper by changing the medium it had grown in. We thus learned that the medium in which the yeast grows is an important parameter that must be taken into account when engineering novel yeast strains to accomplish certain tasks. A reduced medium, as we noticed, although needed for selection of the transformants, can reduce certain abilities of the organism. We can thus try to think once more about what we could change to improve our engineered yeast and make it work better than the wild type. This is where a second round of the engineered cycle starts with a new design phase.

Design

Having learned what we have from these testing and learning steps, we can now think about what to change to make our current system work better in the future. These new decisions must be based on what we have discovered. We have found that our engineered yeasts were not better than the wild type yeast in retrieving copper but at least could absorb copper in the right medium. We also know, from the immunocytochemistry results, that the protein expression is not 100% efficient in a sample of monoclonal transformed yeast. We could try to select positive cells by Fluorescence Activated Cells Sorting (FACS) and have a sample with 100% of cells expressing the protein on their surface to test whether it would better retrieve copper than wild type. If it does, then we prove our point that making yeast express the copper metallothionein at its surface improves its capacity to absorb copper. If not, we should change our strategy. This could be because bio-adsorption is not the solution to our problem and, maybe, digging into bio-accumulation, the intracellular intake of copper could be an idea. A more complete list of our future prospects ideas can be read on the Discussion section.

In addition to this engineering approach in designing our biological system, we went through many cycles in the implementation and hardware design of our project. The details can be found on the Implementation page.

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