Team:USP-Brazil/Safety

Safety forms were approved on September 22, 2013 by Evan Appleton

Overview

1. Would any of your project ideas raise safety issues in terms of:

• Researcher safety
• Public safety
• Environmental safety

Measures were taken to avoid or minimize risks related to biosafety. Figure 1 from the article "Evidence-Based Biosafety: a Review of the Effectiveness Of Principles and Microbiological Containment Measures" by Kimman et. al. illustrates, in summary, the total context of biosafety measures. According to the authors, it is necessary to establish all the risks related to both: work with GMOs (genetic modified organisms) and work with wild organisms. Then, organisms must be classified according to risk classes (1 - 4), and then the measures of physical and biological containment should be taken.

Figure 1: Complete context of biosafety measures. Adapted from the article: “Evidence-Based Biosafety: a Review of the Principles and Effectiveness Of Microbiological Containment Measures” from Kimman, et. al. (2008).

The biosafety level of each laboratory is defined by the organism’s risk class being used. Experiments involving GMOs should be conducted to further characterize the risks related to biosafety (Laboratory Manual Biosafety). Also according to the Manual prepared by the World Health Organization, all analyzed factors should assist in determining the level of biosafety and containment systems required.

According to Kimman et al. (2008), about physical restraints, "further protection of the operator and the environment is provided by physical barriers that prevent or minimize escape of microorganisms from the working place and laboratory." See our product physical container design…

Also according to Kimman, a step subsequent to minimize risks associated with the handling of microorganisms, natural or modified, would be to reduce the ability of the host virulence, use self-inactivating vectors or destruction mechanisms of the organism, which are called biological containment.

All organisms used for our experiments are non-pathogenic organisms, well studied and established (Table 1). These organisms are classified as risk class 1, being characterized as organisms that do not cause disease to humans or animals, according to the criteria of the Brazilian National Technical Commission on Biosafety (CTNBio). This Commission is responsible for assist technical decisions and advice to the Federal Government in the legislation, updating and implementation of the National biosafety manipulation rules on GMOs in Brazil.

Table 1: Description of the organisms used in the project.

Organism	Description	Biosafety level
Escherichia coli	Escherichia coli is a gram-positive bacterium commonly used in research laboratories, due to its rapid growth rate, simple nutritional requirements, well-established genetics and genomic sequence. For the experiments we used the strain DH10B.	Level 1
Pichia pastoris	Pichia pastoris is a methylotrophic yeast, belongs to the phylum Ascomycota. It is a useful system for the expression of gram-to-milligram quantities of proteins for basic research laboratory and industrial manufacture. For the experiments we used the strain GS115.	Level 1

The parts used in our project correspond to promoters, reporter genes and transcription factors, all from organisms class 1 risk, which no represent significant risks to human health. In Table 2, it is possible to view the list of BioBricks used in the project. In that table, are present BioBricks already registered as well as new BioBricks. The plasmids used are also listed.

Table 2: Description of biobricks used in the project. That includes the new biobricks, as well as, the ones already described in the Registry.

BioBrick	Description	Species of Origin	Security level of the species	Origin
RFP	Red fluorescent protein, a reporter protein. This part is described in the Registry, with the code BBa_E1010	Discosoma striata	Level 1	Registry of Parts
Codon-optimized RFP	Red fluorescent protein codon-optimized for expression in Pichia pastoris. This part was submitted by our group to the Registry with the code BBa_K1078001.	Discosoma striata	Level 1	Synthesized, Life.
PAOX1	It is a strong promoter directly controlled by carbon source in P. pastoris. It is activated by methanol, whereas glucose, ethanol and glycerol act as repressors. The PAOX promoter is found three times in the registry of parts; Part:BBa_K431007, Part:BBa_K945000 and BBa_I764001; it is use for heterologous protein expression in P. pastoris.	Pichia pastoris	Level 1	Synthesized, Life.
Modified PAOX1	The promoter PAOX1 modifications makes this promoter more stronger that the wild-type promoter to methanol activation. Submited by our group in the Registry as BBa_K1078001.	Pichia pastoris	Level 1	Synthesized, Life.
PFLD1	PFLD1 promoter is present in Pichia pastoris and can be induced by methanol as a carbon source, but also for methylamine as nitrogen source. After activation by methylamine or methanol is able to obtain expression levels similar to the levels induce by PAOX1 promoter. This part is described in the Registry as BBa_K945001.	Pichia pastoris	Level 1	Synthesized, Life.
Modified Mxr1	Mxr1 (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. Our submitted part is mutated in order to not be repressed by ethanol, as the original Mx1r is. The submitted code is BBa_K1078000.	Pichia pastoris	Level 1	Synthesized, Life.
Plasmid pPIC9K	The pPIC9K vector confers kanamycin resistance for in vivo screening of multiple or single copy inserts. pPIC9K is functional in Pichia strains GS115 and KM71.	Pichia pastoris	Level 1	Synthesized, Invitrogen.

Additional risks involved in this scenario are related to the experiments and chemicals used during the experiments, such as ethidium bromide. This is used as a marker for visualizing nucleic acids in gel electrophoresis. This chemical is mutagenic and must be stored, handled and disposed in a proper way, avoiding risks to people who work with this substance.

According to the Laboratory Biosafety Manual, proposed by the World Health Organization, the “education and training of all personnel involved in the experiments is essential to the effectiveness of all containment measures”. Also according to the manual, the safety measures of laboratory techniques can be compromised by poorly applied or by human error, so the team's commitment to laboratory biosafety rules is essential to prevent accidents.

All participants who worked in the GaTELab and the LabBioTec received instructions on the rules of the laboratories, as well as, training for developing the experiments safely. All members with no previous experience were supervised by more experienced members of the group while developing the project, this aimed to improve the learning experience and as a safety measure.

Figure 2: Biosafety training day at GaTELab.

When we focus on risk to the environment, we must take into account the risks related to possible leaks of genetically modified organism into the environment. Thinking about the worst-case scenario, we cannot ignore the HGT (horizontal gene transfer) and its importance as a mechanism of natural variation among bacteria (POPA and Dagan, 2011).

Prokaryotes, according POPA and DAGAN (2011), have the ability to acquire DNA from the environment or bacteria, and incorporate it into their genome, process called HGT. The special attention given to this phenomenon refers to the fact that the HGT acts as "a source of natural variation, it facilitates the emergence of novel infectious diseases through the spread of virulence mechanisms" (POPA and Dagan, 2011).

In this context, the fact of using for our project the plasmid pPIC9K, which has two antibiotic resistance genes (used as selection markers during the experiments) becomes relevant. The use of resistance genes should be well monitored, minimizing the chances of such genes being incorporated by other bacteria, which can lead to the creation of super bacteria. (WRIGHT, et. al., 2013).

Genes acquired through HGT, are transferred by a range of genetically mobile elements such as: plasmids; bacteriophages; transposons among others ( JUHAS, 2013). There are numerous ways in which the transfer of genetic material between the microorganisms may occur, among the most studied are: conjugation, which consists of DNA transfer by means of plasmids in a process mediated by protein and that necessarily involves contact between cells (POPA and DAGAN 2011); transduction, the genetic material is transferred by the action of bacteriophage and transformation, process in which the cell captures DNA from the environment (JUHAS, 2013). (Figure 2).

Figure 3: This figure illustrates the three most studied mechanisms of horizontal gene transfer. Taken from the article: “Antimicrobial-resistant bacteria in the community setting” from E. Y. Furuya and F. D. Lowy, 2006.

Torres et. al. (2003) notes that “horizontal gene transfer is a source of concern when genetically engineered micro-organisms are intended to be released in large quantities to the environment for bio-technological applications”.

Thinking about taking our biosensor outside the laboratory, we developed the idea of ​​a safety device, which act as physical constraint of these organisms when handled by people outside the laboratory. The device (Figure 3) consists of a pen-like structure made of waterproof transparent material, divided into three compartments, initially isolated one from each other. The lowest compartment is for collecting the drink sample. The second corresponds to the GMOs container. The result is measured by a red staining, when the drink is contaminated by methanol. The third compartment hold bleach for product disinfectant after use.

Figure 4: The Device. (1) The sample is collected in the first slot , the sampler end should be rotated resulting in the closure of the device. (2) The second compartment is released by pressing the middle-part of the sampler, this put in contact the sample with the GMOs. (3) Check the results and press the third compartment (4), releasing the disinfectant material on the GMOs-sample mix. Once closed the device cannot be opened again. Click on the image for more information.

The infographic below (Figure 4) summarizes the risks involved in each stage of the project, which involves the manipulation of GMOs within the laboratories and possible commercialization of this biosensor, which would put them in contact with the public.

Figure 5: Infographic summarizing the risks related to the project.

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

The experiments were developed in laboratories belonging to two institutions of the University of São Paulo (USP): The USP Institute of Biosciences (IB-USP) and the Faculty of Pharmaceutical Sciences USP (FCF-USP). These institutions, have Internal Biosafety Committees (CIBio), which are committees responsible for monitoring laboratories where experiments with genetically modified organisms are developed and also report any accident to the CTNBio (National Technical Commission on Biosafety). All internal committees at each institution, which works with GMOs within USP, are under CTNBio regulation.

CTNBio has been established as part of the federal law No. 8974, of January 5, 1995, also called Biosafety Law, which regulates the use of genetic engineering techniques and the creation of CTNBio. National guidelines for working with GMOs were established by the Ministry of Health. We consulted people related to CTBio, to get information about rules and guidelines on biosafety. The work in the laboratories are under supervision of the head researchers: Dr. Marie-Anne Van Sluys, P.I at GaTE-Lab, established in the department of botany of the IB-USP and Adalberto Pessoa Junior, chief researcher of the laboratory LabBioTec at FCF-USP.

3. 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?

When we analyze our current project, we found a hypothesis that stand: GMOs will be produced on an industrial scale for commercialization. Moe-Behrens et. al (2013) confirms this scenario with the quote: "if speculations correctly predict the future use of synthetic biology, the technology will scale to large industrial volumes"; this brings the following concern: how to generate a biological containment mechanism that is indeed effective in improving safety in synthetic biology open systems?

Scientists have already studied this concern. One of the most studied strategies is the use of auxotrophic, according to Moe-Behrens et. al (2013) this biocontainment method would work as follows: genes that synthesize compounds essential for the organism would be disabled, and such compounds would be available in the medium. If the organism escapes, it will be unable to synthesize these essential compounds, which will lead to its death. Figure 5B illustrates this mechanism.

Another technique is inducing lethality (Figure 5A), in which the organism survives thanks to a substance that is added to the medium that repress the expression of a toxin that would kill the cells. Out of the selective medium the toxin will be express. This system, as well as others not mentioned here, is based on the toxin-antitoxin system. “The activity of a small toxin (<15 kDa) is stopped by a short-lived, cis-encoded antitoxin” (WRIGHT, et. al., 2013). There are three types of systems, namely: the type I antitoxin, is an antisense RNA that inhibits translation of the toxin; type II, the antitoxin is a protein, and finally, the type III antitoxin is an RNA molecule that attaches and inhibits toxin directly. (Wright, et. Al., 2013).

Figure 6: Illustration showing biosafety devices. A compound provides control of the microorganism, because it inhibits expression of the toxin gene (A) or because it is essential to nutrition since the gene that synthesizes the compound was deleted (B). Scheme taken from the article "Preparing synthetic biology for the world." Moe-Behrens et. al, 2013.

Our idea, for our future iGEM projects or for improving the biosafety of our current project, is to use natural biological systems already described in bacteria (like the toxin-antitoxin system) and modify them to serve as an intrinsic safety mechanisms to prevent that synthetic organisms may escape into the environment. This is a future improvement of our current physical container approach.

References

Torres, B., S. Jaenecke, K. N. Timmis, J. L. García, and E. Díaz. 2003. A dual lethal system to enhance containment of recombinant micro-organisms. Mycrobiology. 149: 3595-3601.

Popa, O. and T.,Dagan. 2011. Trends and barriers to lateral gene transfer in prokaryotes. Curr. Opin. Microbiol. 14: 1-9.

Juhas, M. 2013. Horizontal gene transfer in human pathogens. Crit. Rev. Microbiol, Early Online, 1-8.

Moe-Behrens, G. H. G., R. Davis, and K. A. Haynes. 2013. Preparing synthetic biology for the world. Frontiers in Microbiol. 4: 1-10.

Wright, O., G-B. Stan, and T. Ellis. 2013. Building-in biosafety for synthetic biology. Microbiol. Papers in Press. Microbiol. 159: 1221-1235.

Kimman, T. G., E. Smit, and M. R. Klein. 2008. Evidence-Based Biosafety: a Review of the Principles and effectiveness of Microbiological Containment Measures. Clin. Microbiol,. Rev. Vol. 21 3: 403-425