We have our own iGEM TU Delft lab in the new Applied Science building on the edge of the TU Delft campus. It is classified as an ML-1 lab, the lowest safety level to work with modified organisms, which is sufficient for our experiments. In this lab we do all the molecular work, such as cloning, PCR and transformations. For characterization work, such as measuring fluorescence or growth curves, as well as growing our cells we can make use of the equipment of the Department of Bionanoscience of the university.
Since our custom-built setup involves working with lasers, we need to have a specific optical lab for this. For both our safety and the accuracy of experiments, this lab is completely light-isolated, no light can go in or out. This lab is also classified as ML-1, so we can bring our organisms inside. The lab has all the requirements for laser safety, such as a warning sign and an interlock that will shut everything down when the door is opened or in case of emergency. Apart from this lab, we are also working in other microscopy labs of the Department of Bionanoscience, which have the same setup and safety regulations.
Our office is our home for when we're not working in the lab. Here we work on parts of the project such as the safety tool, the wiki, the modeling, and processing our results. This summer the Bionanoscience department moved to a new building, so it took some time before we had our own office. When we finally got our office, we quickly made it our home. Next to our office, there is a meeting room, where we have a weekly meeting with our TA’s and PI’s to keep everyone up to date and discuss problems we might encounter.
General Lab Safety
Our lab is classified as Level 1 of biosafety. This is the lowest safety level, meaning that our experiments involve low or no risk. Therefore, we only handle organisms of Risk Group 1, these do not cause any disease in healthy adult humans. The biological materials used for our experiments are handled in an open bench. All the members of the team have received safety training and tests, including:
- Introduction to sterile working
- ML-1 safety training + test
- Chemical safety training + test
- General safety information, regarding contact persons and locations + test
The safety of our experiments was supervised by Susanne Hage (wetlab coordinator of the department of Bionanoscience of the TU Delft) and Marinka Almering (Biosafety officer of the Faculty of Applied Sciences TU Delft). The research has been conducted with respect to the regulations of biosafety for The Netherlands, that can be found here.
Before starting any experiments we have written a research proposal including all our strains, DNA fragments, chemicals and experimental setups that we planned on using, as well as safety precautions, mini-MSDS’s and disposal requirements. This research proposal was approved by the biosafety officer prior to starting the experiments. The full research proposal can be found here.
General Laser Safety
Since working with microscopes involves working with lasers, we all received basic laser/microscopy safety training. Furthermore, the team members that were involved in building the optical setup for the hardware component of our project also received an advanced laser safety course prior to their work in our optical lab. The laser safety of our project was supervised by Jeremie Capoulade (Head of the microscopy facility of the Department of Bionanoscience).
Safety of our project
For our project we make use of the host organism Escherichia coli, a harmless organism of Risk Group 1. The organism is transformed with a gene to express Silicatein-α, originating from two species of sponges, T. aurantia and S. domuncula. This enzyme will enable an organism to cover itself in a coat of polysilicate. E. coli is only able to produce the enzyme upon transformation of our designed plasmid and after induction with IPTG. Furthermore, our cells are also able to fluoresce after transformation with three different types of fluorescent proteins, GFP, mVenus and mKate. These proteins are expressed constitutively. We also have investigated native E. coli genes that might influence cell shape. The design of our parts is based on several scientific papers and previous iGEM projects.
Since we use genetically modified bacteria that have gained new functions compared to its wildtype, we have to be very careful in following biosafety guidelines. The properties of our bacteria, synthesizing glass and fluorescence, are not directly harmful to humans, but these properties in bacteria haven't been observed in nature before, so we don't know what the effect of these bacteria on the environment could be. Therefore, we are very careful in following strict ML-1 rules to make sure the bacteria stay in our lab, which includes sterilizing waste, washing hands and keeping lab material, such as lab coats, within the lab. The work space is sterilized with ethanol before and after working with our bacteria. We also use some harmful chemicals, such as ethidium bromide, Silicic acid hydrate, Tetraethyl orthosilicate, Rhodamine 123 and Sulphuric acid. These chemicals are handled with care wearing the right protective materials and clothing, following de MSDSs in our research proposal, see the paragraph ‘General lab safety’. Team members with little to none experience with handling GMO’s were under supervision of instructors and team members with at least one year of experience.
For our project we have used the E. coli K-12 strain TOP10 for cloning, transformation and proliferation of our designed BioBricks. Upon confirmation of the correct cloning of our BioBricks in TOP10 we purified the BioBricks and transformed them into our chassis organism, E. coli B, strain BL21. All characterization experiments were carried out using this organism.
To evaluate the safety of our project, we have developed a safety tool,: an online software tool that can evaluate and qualify the safety of our project, as well as the safety of other synthetic biology projects. More information on our safety tool can be found here.
Environmental impact of our project
At the current stage of our project, our cells are maintained in the lab and do not cause any risk to the environment at all. However, our biological lenses are eventually destined for products that can be used outside an ML-1 lab, e.g. in solar cells, cameras or microscopes. The only real risk that our project might pose is that the bacteria are encapsulated inside the glass spheres; this means that the lenses we produce contain a core of a live bacterium. Current (European) ML-1 safety regulations do not always allow the use of GMO's outside the lab. Besides that, it is safer to have a product that does not contain any bacteria at all, in that case, if the glass will break it won’t cause any contamination. Therefore, we need to find out how to get the bacteria out of the biological lens, without distorting its optical properties. We are currently doing literature research in how to metabolically stop a cell, without breaking it down completely, since the cytosol is suspected to contribute to the optical properties of the lens. Firstly, we are testing if the cells are still able to grow in their silicate layer, if this is not the case, we don’t necessarily need to take any extra measures before implementing our product in the environment. However, mutations could occur so it’s always safer to make sure the bacteria are not able to reproduce anymore. Therefore we are also experimenting with autoclaving, freeze-drying and UV-sterilizing our cells to see if this kills them without disturbing the optical properties. More information can be found on our projects page.