Contents 1 Biosafety proposals 1.1 Killer gene 1.2 Susceptibility Construct 2 Biosafety Questions

Biosafety proposals

WHY Biosafety?

All people need to feel safe. Instinct drives behavior; therefore no technology will have a widespread use if safety is not guaranteed. This issue has been addressed by several research groups, however no Biosafe Biobrick standards have been defined within the iGEM community.

We strongly believe that biosafety is not an option but a fundamental requirement regardless of the intrinsic risk of the DNA circuit.

Under this vision, we have conceived Two main systems, one is inspired by nature, easy to use, cheap and extensible to all gram negative bacteria that -if working properly- we urge to be introduced mandatory to all plasmid backbones in the registry.

The second system is an integrative plasmid that knock out a gene necessary for copper stress tolerance.

Killer gene

To our vision a reality we propose the inclusion of a short and simple lysis gene under a constitutive promoter in all biobrick plasmids. This sequence codes for the mE gen phi X174 phage which inhibits peptidoglycan biosynthesis(Ref).

In this way it would kill any gram negative bacteria unless grown at all times under higher magnesium sulfate concentrations than those found in nature. In consequence, cells expressing the gen would be unable to thrive in an environment other than strict laboratory conditions.

Description : Although the mechanism for which MgSO4 interferes with the mE gene is not known, when MgSO4 concentrations of 0.2M are present, the mE gene does not exert any deleterious effect on bacteria.

Our next goal is to test this biosafety mechanism by assembling a model construct to transform E. coli in order to characterize its effectiveness.

Susceptibility Construct

Biosafety Questions

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

No pathogenic microorganisms nor dangerous genes are considered in our ideas, in consequence our intended projects, if executed, do not represent any risk to researchers nor public safety.

• environmental safety?

As Synechocystis is a naturally competent environmental bacteria that undergoes homologous recombination, there is a posibility that if laboratory recombinant strains are leaked to the environment there may be environmental safety issues such as lateral DNA transference and endogenous species endangerment. We discuss this issue further in question number 4 (see below).

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

Yes, the release of biobricks made from Synechocystis genome´s sequences or at least with a region homologous to it raise the possibility of recombination by environmental cyanobacteria. To address this issue we have proposed and designed a biosafety mechanism discussed in question number 4 (see below).

Furthermore, our laboratory practices enforce strict methods to handle any biological material. As DNA is always hermetically contained or discarded with other biological material according to standard protocols we believe that our practices will not produce any environmental hazard.

• did you document these issues in the Registry?

All issues regarding our biobricks will be documented once we send the parts to the registry.

It has to be stated that any of the sequences designed and/or handled by our team are not per se dangerous nor they represent a fitness advantage to recombinant strains.




• how did you manage to handle the safety issue?

Our lab follows the Manual of Biosafety, established by the Superior Counsel of Science and Technological Development, from the National Fund of Scientific and Technological Development of Chile (FONDECYT).




Click here to download the Manual of Biosafety (Spanish)

• How could other teams learn from your experience?

To adress the possibility of recombinant Synechocystis cells being released from the lab, we've designed a recombination plasmid that knocks out the copS gene. This gene codes for a Cu-binding protein and is essential to Cu stress response in Synechocystis. It has been demonstrated that strains lacking this gene can´t survive in much lesser Cu concentrations found in drink water or natural water bodies (2).

We advice every team working in this chassis to adopt similar strategies. By designing integrative plasmids which interrupt fitness related genes, recombinant strains will be auxotrophic and/or hiper-susceptible. Thus, their recombinant cyanobacterial strains will be unable to thrive in natural environments.

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

Yes, there is a local biosafety panel in our institution. The Comitee of Bioethics and Biosafety of the Biological Sciences Faculty of the Pontifical Catholic University of Chile has reviewed our work and determined that the procedures involved in our project follow the bioethical and biosafety standards for research regulations provided by the Chilean Comission of Scientific and Technological Research (CONICYT).

Click here to download our Certificate from the Comitee of Bioethics and Biosafety




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?

To prevent future problems and to encourage the construction of biosafe standard biological parts, we, UC_Chile team, propose the creation of the section “biosafety analysis” mandatory for all parts before their shipping to the registry. This way, it is assured that each new biobrick or device includes an assessment of the risks and biosafety advantages involved in its physical implementation.

Alongside the previous, we suggest the characterization of biosafety levels of other team’s constructs, making this practice analogous to the existing standard characterization of bricks. The requirement of this biosafety assessment for a team to win the gold medal would be a strong incentive.

Regarding to the molecular methods by which biosafety could be improved, we believe that the implementation of a lethal system to all iGEM plasmid backbones would prove essential. The system would kill the recombinant cells unless its action is inhibited by a compound present in the culture media but that its concentration in the environment is insignificant. That way, recombinant cells which are leaked to the environment will have no possibility to thrive in environmental conditions. (Check details above!)