Background / Research

Bioinformatics - building a multiple metal binding protein

At the beginning, we started an extensive research to find suitable sequence-parts for our own heavy metal binding protein (T4MBP) and the cellulose binding domain (CBD). The consideration of the sequence origin is an important aspect for biosafety reasons at the beginning of every project. Therefore, we studied reports in large scale to get impressions about studies, experiments and insights of these genes. With these information, we were able to estimate the properties of metal binding for these proteins.

We found four metal binding amino acid sequences and a cellulose binding domains. Our overall goal was to obtain a transgenic plant expressing the GOIs. In a next step, the codon usage was adapted by reverse translation of the corresponding amino acid sequences into DNA sequences under consideration of the codon usage (Escherichia coli and Arabidopsis thaliana). Simultaneously, we eliminated unwanted sites via silent mutation. Before sequence assembly, we inserted one Serin-Glycin Linker between each part and the CBD. To suppress RNAi effects, the codon usage was altered by silent mutation.

To predict the starting point of translation, we inserted our combined insert into the vector and used bioinformatic tools. Caused by the regimentations of iGEM, we removed all forbidden restriction sites. As a final step, we integrated a highly efficient 5´UTR to enhance expression.

To look over the rim of the tea cup

When we finally decided on „Plant against“, the invention of a protein that is able to bind heavy metals, as our topic for iGEM 2014, we also thought about the impact our project might have. Quite early we recognized that there is no chance of designing a useful gadget in one year because no preliminary work was done so far. So, we really had to get it started from the very beginning and of cause, wanted to test our bioinformatically evaluated construct in E. coli and plants such as Arabidopsis thaliana and Nicotiana tabacum to lay the foundation for an application in terrestrial and aquatic plants. The main question that we bore in mind concerned the impact on the environment: What problems do heavy metals cause and how could our protein help to solve them? To answer this question we firstly did a lot of research concerning urgent problems and impacts of heavy metals. After all this theoretically stuff, we decided to go into practical research and visited the sewage works, Herrenhausen (Hanover, Germany). There we had a nice talk to some employees and they helped us quite a lot to distinguish the problems from the chances we were going to have with our project.

Within the project, we always conferred with people who are working in fields where heavy metals are a big problem for the environment (e.g. one of our sponsors) as well as e.g. university working groups (e.g. Research Group Analytical Chemistry) that aim to quantify the amount of heavy metals in the environment by using special analytical methods such as ICP-OES. Furthermore we discussed the topic with passers-by in Hanover’s pedestrian area at SynBio Day. Now, at the end of 2014’s project, we can say that we got a good impression on problems concerning heavy metals in environment and that we may have paved the way to solve some of them by inventing and successfully testing our T4MBP. Maybe next year we come up with a useful gadget…Who knows…

Our endeavor to fullfill medal criteria

On this page you get an overview about our attempts to fullfill medal criteria.