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From 2012.igem.org

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

   No pathogenic microorganisms or dangerous genes were part of 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, the idea of genetically engineer it raises environmental safety issues regarding the acquisition of biobricks by other cyanobacteria outside the lab.

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, Yes, the release of biobricks made from Synechocystis genome´s sequences or at least with a region homologous to it raises the possibility of recombination by environmental cyanobacteria. As DNA is always hermetically contained or discarded with other biological material according to standard protocols, there´s little chance for it to happen.
   • did you document these issues in the Registry?

   It has to be stated that any of the sequences designed and/or handled by our team are per se dangerous nor they represent a fitness advantage to recombinant strains. Regarding to living cells, as they carry recombinant DNA, the same precautions as for that molecule apply.

   • how did you manage to handle the safety issue?

   Recombinant and non-recombinant cells are exposed to a heat shock (80°) previous to ethanol washing and discard in biosafety cabinets Our lab followed 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).(1)

   • How could other teams learn from your experience?

   For adressing the possibility of recombinant Synechocystis cells being released from the lab, we 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 demosntrated that strains lacking this gene can´t survive in Cu concentration much lesser than found in drink water or natural water bodies.(2) We advice Every team working in this chasis to adopt the strategy of designing integrative K.O plasmids in order to develop auxotrophic and/or hiper-suceptible strains unable to trhive in the enviroment.

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

Yes, in our institution we have a local biosafety panel. The Comitee of Bioethics and Biosafety of the Faculty of Biological Sciences 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 on Scientific and Technological Research (CONICYT).

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

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

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. In this way, 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 addition of a “bomb” gene to all iGEM plasmid backbones could be very useful. This gene would kill the recombinant cells unless its action is inhibited by a molecule present in the lab culture media but with an insignificant concentration in the environment. (1)

The best candidate we found is the phiX1714 phage lysis gene E. When placed under a constitutive promoter, this gene inhibits peptidoglycan biosynthesis and therefore lyses the cells (2). It has been shown that the addition of aprox. 0.2 M MgSO4 to the medium (much less than the amount founded in common water) prevents cell death by the gene E (and lysis in general) even when its expression has already been induced (3).

To ensure cell lysis efficiency the optimized mutant gene mE, which produces lysis in 99,999% of cells (4), should be used.


Footnotes

(1) Leduc M, Kasra R, van Heijenoort J. Induction and control of the autolytic system of Escherichia coli. J Bacteriol. 1982 Oct;152(1):26-34. PMID: 6181050 [PubMed - indexed for MEDLINE]

(2) Young KD, Young R. Lytic action of cloned phi X174 gene E.J Virol. 1982 Dec;44(3):993-1002. PMID: 6294347 [PubMed - indexed for MEDLINE]

(3) Gschwender HH, Hofschneider PH. Lysis inhibition of phi-X174-, M12-, and Q-beta-infected Escherichia coli bacteria by magnesium ions. Biochim Biophys Acta. 1969 Oct 22;190(2):454-9.

(4) Yu SY, Peng W, Si W, Yin L, Liu SG, Liu HF, Zhao HL, Wang CL, Chang YH, Lin YZ. Enhancement of bacteriolysis of shuffled phage PhiX174 gene E. Virol J. 2011 May 6;8:206.