- chose a non-pathogenic chassis
- chose parts that will not harm humans/animals/plants
- worked under the existing gene technology safety regulations, in an appropriate lab environment and with appropriate lab
- recommend including an "induced lethality" or "kill-switch" device for future use
Would any of your project ideas raise safety issues regarding researcher safety, public safety, or environmental safety?
Our Labwork is constantly overseen by Ph.D. students, postdocs, and professors of our institute, the Interfaculty Institute of Biochemistry. All of them are very experienced in laboratory work, with expertise from genetics over molecular biology to structural biology. To ensure replicability, we document all of our work in our laboratory notebook. Our Team follows widely used laboratory protocols in an appropriate laboratory facility (biosafety level 1 laboratory, S1), as requested by German law ("Gesetz zur Regelung der Gentechnik (GenTG)," German Gene Technology Law and “Verordnung über die Sicherheitsstufen und Sicherheitsmaßnahmen bei gentechnischen Arbeiten in gentechnischen Anlagen (GenTSV)”, Genetic Technology Safety Regulations Act) to guarantee the safety of the researchers and the public.
The participation in regular meetings about laboratory safety is an essential element of the education at the University of Tuebingen. Additionally, all of our lab members were specially instructed about biosafety. The organisms used in our project, namely Escherichia coli, and Lactobacillus Johnsonii, are not only well-known and considered safe, they even occur naturally in the human intestine. Due to the absence of any pathogenic, toxigenic, or colonizing activities in the organisms and the harmless nature of our parts, there are no safety concerns during our work. Furthermore, the transformed Lactobacillus is nonviable outside a fructose-rich medium, which is another convincing fact regarding the absence of security concerns. In the case of success with the Lactobacillus, we would have tested our system with fructose-intolerant yeast, which has the same safety properties as E. coli and L. johnsonii. We do not plan to experiment with any other organisms. Our project is intended to be used in the laboratory. We are working at an S1 laboratory, which means there are several methods and guidelines to ensure the safety of researchers and the environment, but it limits research to well known and nonhazardous species.
What is the goal of the project? What will the engineered organisms do?
The goal is to make the first step to fighting fructose intolerance. The engineered Lactobacillus will be able to uptake great amount of fructose, thereby reducing the fructose level of the surrounding. To accomplish that, we will overexpress the phosphofructokinase to allow a faster degradation of fructose. Glucose as a prime energy source of the cell will be sealed off by knocking out the glucose-6-phosphate isomerase and the glucose 6-phosphate dehydrogenase, thus leaving the Lactobacillus dependent on fructose. Transporters for sucrose and fructose will be overexpressed to ensure the energy supply of the cells and to drain the surrounding from all fructose.
What risks does your project pose at the laboratory stage? What actions are you taking to reduce those risks? Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?
All organisms we are using in our project are very well characterized and not known to pose any risk or danger to humans or the environment. This also applies to the BioBricks used in our system. According to the German Gene Technology Law, the bacteria, yeast, and recombinant DNA components are classified as S1 according to the German Gene Technology Law (GenTG). Our experiments are conducted under S1 conditions, which implies a lot of safety features. These conditions include the usual safety features of wearing rubber gloves and laboratory coats, sterilizing waste, regularly disinfecting the workspace, etc. The BioBricks designed by our team are not found toxic or dangerous by any means. None of the parts we use are known to have any negative or harmful effect on humans or other organisms.
How would your project be used in the real world?
Our project could be used for probiotics: A patient with fructose intolerance could take up the lactobacillus via a probiotic product (e.g. a yogurt) together with a fructose-containing meal. In the intestine, the Lactobacillus would digest the fructose, preventing negative side effects for the patient. After the fructose is degraded, the Lactobacillus, incapable of digesting glucose, would lack further carbohydrate sources and die off. However, lots of further research would be necessary to get to this application, especially as the Lactobacillus is a GMO.
What risks might your project pose, if it were fully developed into a real product that real people could use? What future work might you do to reduce those risks?
Since the Lactobacillus is a naturally in the intestine occurring bacteria and no new functions were introduced, the risk for the patients can be considered very low. Furthermore, the Lactobacillus can only live in a fructose-rich environment, which prevents uncontrolled spreading. To further ensure the prevention of spreading, we would recommend additional kill switches which detain the organism from living anywhere else except the desired environment. A full proofed safety concept would be a central part of any further research on an application of our approach.
Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices, and systems be made even safer through biosafety engineering?
Promoters that are activated in an uncontrolled environment or anywhere outside the laboratory conditions could express parts that can induce cell death via apoptosis or autolysis. A vast amount of different examples for so-called kill switches was designed by different teams of the iGEM community and is now available in the registry (see for example 2011's team of UCL London and the 2013’s BGU Israel's iGEM-team "Programmable Autonomous Self Elimination").