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 protein-based micro compartments as a new in vitro technology for science. The idea is going to be developed at the laboratory with the proper biosafety conditions of handle and disposal of reagents and biological material. For those reasons it does not represent any public and/or environmental biohazard.

In addition, our project involves the use of microorganisms with a biosafety level 1 (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). Also, the BioBricks or genes used in this chassis do not show any potential biological hazard. The utilization of antibiotic resistance as selection markers could be the only potential bio risk.

Our laboratory has a well documented and stabilized protocols to dispose any biological material and to discard different reagents with environmental toxicity. The reagents and others are discarded after a particular number of decontamination processes as autoclaving, chemical disinfection, and incineration (3). Therefore, the environment should not be affected by our work. This guideline of biohazard disposal is part of the regulation from the National Commission for Scientific and Technological Research (CONICYT) in Chile.

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

As we described above, the biobricks and/or genes used in this project do not represent any potential biological hazard.

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

Yes, there is. The Committee of Bioethics and Biosafety of the Biological Sciences part of the Faculty of Pontificia Universidad Católica of Chile. This committee is responsible for ensuring good practices in the laboratories in our university and also, 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 to 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 pipeline 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. Additionally, another important point in the global discussion on GMOs such as the effect of synthetic MO in the native organisms could be avoided. As we describe above, we hope to reduce the use of living organisms and all complications in terms of biosafety that entails.

Our project has another relevant feature: an autolysis system. It meets two very important functions, one of them is helping the extraction of BMCs and the second one is that it is safety way to eliminate the microorganisms that we are using. With those functions, we are making sure that it will not be dispersed to the environment.

Click here for more information about the autolysis device.

Other idea that came to our minds in a certain point of this project development is the generation of bricks with removable resistance. This could be achieved using an invertases-based system in which the invertases come from phages. Thus, we would generate deletions in the DNA regions with the resistance and later switch the resistance locus with a new marker like a a fluorophore or chromophore.

Through this simple strategy, Synthetic Biology could face one the main drawbacks of not-target strains with undesirable antibiotic resistance. It is well known and documented that some laboratory bacterial strains are antibiotic resistance and they represent environmental risks because they could transfer the resistance to other pathogenic bacteria or other unwanted organism.

Our idea is inspired by the somatic recombination of the immune system. This immune strategy is based in the principle where RAG-1 and RAG-2 recognize specific segments called Recombination Signal Sequences (RSSs). The 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.
In our journey to find proteins with new features, we found the Marburg project in 2012(4). This project proposes the use of an invertase from Phage Mu to generate random deletions and thus create diverse phenotypes. The invertase in the phage recognized the GL (gixL) and GR (gixR) sites, inverting the called G-segment that contains two genes in which those have been expressed by the same promoter but with a different conformations.
In simple words, our idea is looking forward to a have constitutive expression of a resistance cassette to use it as a selection marker in transformations but the design of the construct implies that the resistance in the CDS will be flanked by Gl and Gr. Those regions are going to be recognized by the invertase enzymes. Therefore, when the induction of the invertase has taken place it will remove that undesirable region from the plasmid and the resistant bacteria will be converted to non-resistant bacteria expressing another reporter protein which represent a less biosafety issue like a fluorophore.

The idea is presented in the following diagram.