1. Would any of your project ideas raise safety issues in terms of researcher, public safety or environmental safety?

Our project proposes the utilization of proteinaceous microcompartments as a new in vitro technology for science, so the idea itself, being limited to the use only within a laboratory, would not generate a public or environmental hazard.
Also, our project only involves type 1-risk microorganisms (Top10, BL21, and Dh5α strains that are E. Coli K12 derivatives) according to the Risk Group Table and the World Health Organization Laboratory Biosafety Manual (1,2); and the BioBricks or genes used in this chassis don’t have any potential biological hazard. The only one is the utilization of antibiotic resistance as selection markers. In our laboratory there are protocols established by the National Commission for Scientific and Technological Research (CONICYT) to disposed any biologic material and different reagents with environmental toxicity, they are discarded after a number of decontamination processes like autoclaving, chemical disinfection, and incineration (3). Therefore, the environment shouldn’t be affected by our work.

2. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?

As described above, the bricks or genes used in this project don't have any potential biological hazard.

3. Is there a local biosafety group, committee, or review board at your institution?

Yes, The Committee of Bioethics and Biosafety of the Biological Sciences Faculty of Pontificia Universidad Católica of Chile, this committee is responsible for ensuring good practices in the laboratories of our university, and this is achieved through the regulations provided by the Chilean Comission of Scientific and Technological Research (CONICYT).

4. 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?

The main use we want to give our project is to generate metabolic reactions within the BMCs, and thus decouple metabolic pathways from organisms and bring them to an in vitro level. This will help to solve biosafety concerns and problems. For example, not using any kind of microorganisms (MO) for those processes will decrease the use of selection markers such as antibiotic resistance cassettes and the lateral gene transfer would be much more difficult; or very important points in the global discussion on GMOs such as the effect of synthetic MO in the native organisms could be avoided. With this, we hope to reduce the use of living organisms and all complications in terms of biosafety that entails.
Yet another important feature that our project has is an autolysis system that meets two very important functions, helping the extraction of BMCs and safely eliminate the microorganisms that we are using, thus making sure that will not be dispersed to the environment.
Click here for more information about the autolysis device
Other idea that came to our minds was the generation of bricks with removable resistance. This could be achieved by constructing a system using invertases from phages. Thus, we would generate deletions in the regions with the resistance, and later confer a new marker, which in this case could be a fluorophore or a chromophore.
This way, one of the biggest drawbacks of Synthetic Biology could be avoided, which is to make laboratory strains of bacteria resistance to antibiotics indiscriminately, creating potential environmental risks by transferring these resistances to pathogenic bacteria or other unwanted organism.
This idea was inspired by the somatic recombination of the immune system, where RAG-1 and RAG-2 recognize specific segments called Recombination Signal Sequences (RSSs), where complex of proteins can invert or remove segments of DNA from the genome depending on the position of the RSSs. The whole process is performed in order to generate diversity in the generation of antibodies.
Image of the two options in Site-specific recombination: Inversion and Deletion.
Looking for proteins with similar characteristics, we found the Marburg 2012's project (4). That project uses an invertase from Phage Mu to generate random deletions and thus create diverse phenotypes. The invertase in the phage works recognizing the GL (gixL) and GR (gixR) sites, inverting the called G-segment that contains two genes, which are expressed by the same promoter but in different conformations.
G-Segment of phage Mu, with the flanking sites GL and GR.
In basic terms, our idea is to have constitutive expression of the resistance cassette to use it as a selection marker in transformations, but the design of the construct implies that the resistance's CDS will be flanked by Gl and Gr, regions recognized by the invertase. So, when the induction of the invertase has taken place, it will remove that region of the plasmid, and the resistant bacteria will be converted to non-resistant bacteria with expression of another reporter protein with less biosafety problems, like a fluorophore.
The idea is presented in the following diagram.