Think before doing
In the Netherlands, the RIVM (the Dutch National Institute for Public Health and the Environment) conducts research to make sure that we keep ourselves and our environment healthy. This also includes opening discussions about the developments in synthetic biology and the safety issues regarding this. More information about the general safety aspects of our project can be found here.
This year the RIVM encouraged the Dutch iGEM teams to 'think before doing': think about how we are going to incorporate safe-by-design in our project.
Safe by design
Below you can find a short description of our implementation of "Safe by Design". If you want to know more about certain topics use the arrows.
What is Safe by Design
Safe by design is a way to incorporate safety in every design aspect: start to think about safety issues and risks even in the design stage of the project to identify the risks beforehand instead of considering them afterwards.
The first step to incorporating safe-by-design is to think about what is meant by safe-by-design. Safe-by-design can be seen as a way to incorporate safety in every design aspect. By starting to think about safety issues and risks even in the design stage of the project, risks can be identified beforehand, instead of considered afterwards. Key to this is to not only to think about the components (DNA, chemicals, proteins) which are used, but also about the outcome of the reactions taking place. For instance, if a chemical reaction could produce unwanted by-products, these should also be considered.
In our project we do not design a bacterial system, but we work with proteins which can form a network. The scaffold protein and its binding partner are well studied at our university. The small molecule inducer is a toxin, but research has shown that it has no measurable negative effects in high concentration. The proteins will be used in our project to make a network, we have modeled this network formation to provide us with more information about this.
For our project we use Escherichia coli as a host organism, which is an organism of risk group 1. This organism is then transformed to express our proteins constructs: a scaffold protein (14-3-3) and its binding partner (CT52). Since Escherichia coli is only used to produce the wanted proteins, we are not developing a bacterial system in which safety mechanisms like a kill switch can be induced. However, we can look at the safety of our proteins and at the safety of the network that is formed.
First of all, 14-3-3 and its binding partner are well studied proteins at our university: the sequence is known and our lab has experience with the expressed protein and optimized its amino-acid sequence for different applications. This eliminates risks present if one would start working with ‘new’ or relatively unknown proteins. For network formation, we will use a small molecule inducer, fusicoccin. Fusicoccin is a toxin produced by fungus and has detrimental effects on plants. However, research has shown that fusicoccin in a high concentration does not show measurable negative effects on mammalian cell lines, so it would be safe to use as an inducer.
Besides the proteins itself, we also thought about the network formation. Since the interaction between 14-3-3 and its binding partner are well known, we have worked on creating a model. This way it is easy to screen concentrations and conditions under which the network is formed, providing us with more information.
The application of our project lies in encapsulating cancer cells. The idea is that an inhibition site is present which prevents the two proteins to interact. However, a cleavage site is introduced which can only be cleaved by proteases excreted by tumor cells, thus making it a specific process.
What if it goes wrong
To prevent unwanted gelation, an extra cleavage site can be introduced, specific for another protease. When the network formation occurs at an unwanted location, this other protease can be added and the network will fall apart.
Rules & Regulations
Since our project involves the use of GMOs (Genetically Modified Organisms) we decided to take a deeper look into the GMO policy in the Netherlands.
Next to GMO policy, we also wanted to investigate the rules and regulations around the application of our project: the encapsulation of tumor cells. Via the RIVM we got into contact with Claudette de Vries, she works for the RIVM at the Centre for Health Protection. This centre focuses on the health impacts and risks to people of chemical and biological agents, with a focus to medicines among other things.