With the increase in the number of people working in laboratories, researchers and laboratory technicians increasingly are exposed to a multitude of biological, chemical, and physical hazards. These include infectious microorganisms, toxic chemicals, and skin lacerations. Moreover, continuous generation of genetically modified organisms (GMOs) or genetically modified microorganisms (GMMs), if released into the environment, can pose unexpected consequences to the ecosystem. As a result, the need for safety in all aspects is of great importance in preventing serious accidents and unwanted risks.
During the development of our project, OCYANO, four aspects of safety were taken into account, which will all be discussed in this section. Firstly, laboratory safety was studied, as our colleagues from the Wet-lab team were exposed to several potential laboratory risks when performing experiments. Mrs. Veerle Verdoodt from the lab of Prof. Van de Poel and Mrs. Marleen Voet from the lab of Prof. Lavigne kindly gave the Wet-lab team members a special laboratory safety seminar before the start of experimental work.
Afterward, we investigated the ethical implications brought by OCYANO onto society, wherein Prof. Pascal Borry generously helped us, resulting in a further look at the legal aspects of our project. Consequently, we invited the European Commission to kindly explain to us the policy and legislation that surround GMOs in the European context. Lastly, we asked the question among ourselves, if our project were to become industry scaleable, what other aspects should we consider to ensure a safe design? We consulted several recent studies for the answer to this question.
When working with the cultures of the fast-growing cyanobacterial strains Synechococcus elongatus UTEX 2973 and Synechococcus sp. WH8109, our iGEM team was allowed to work under the laboratory of Prof. Bram Van de Poel. Please note that the cyanophages were manipulated in a different laboratory, in turn, the related discussion will be separate. Before holding access to the laboratory of Prof. Van de Poel, the Wet-lab team needed to participate in the Health, Safety, and Environment (HSE) training course (abbreviated as VGM in Dutch) led by Mrs. Veerle Verdoodt. Only the Wet-lab team members were given access to the lab as an extra biosecurity measure due to the interdisciplinary nature of our team.
During the HSE course, Mrs. Verdoodt explained the different safety features of their laboratory, the schedule for sterilization or autoclaving of dry and liquid containers, the waste lab management system, emergency protocols, and the proper behavior in the laboratory. Below are the following files she generously shared, concerning the HSE course: wel
- Welcome Brochure,which contains all the general information on HSE of the corresponding laboratory facilities
- Safety Information Document,which contains more in-depth information in case of an emergency such as first aid kits, evacuation routes, and fire alarm signals
More importantly, Mrs. Verdoodt showed us our designated workspace, where the level of biosafety is at level 2. Our workspace, equipped with a laminar flow cabinet, allowed work under sterile conditions when handling the cyanobacterial cultures. As a rule of thumb, we were required to wear a laboratory coat and a pair of gloves while performing experiments. However, when entering the cyanobacterial culture room, it was also necessary to wear additional shoe covers to prevent unnecessary contamination. Near the laboratory, there was also a first aid kit that can be used in case of a small accident (see Figure 1).
Figure 1: The first aid safety kit used in the case of small accidents.
The laboratory included the chemical room that contained different risk classes, E1 (low risk) to E4 (high risk) of chemical compounds. Before using chemical compounds that are at risk class E3 or E4, a risk assessment of the chemical compound had to be submitted to Mrs. Verdoodt. This assessment form was filled out by the safety manager of our iGEM team, who identified the hazard and precautionary statements of the chemical products before sending it to Mrs. Verdoodt. Thus, while handling the chemical product of risk class E3 or E4, additional precautionary methods can then be applied, such as wearing face masks and glasses. These were very crucial for us as we were using toxic chemical compounds in the creation of the SN growth medium for Synechococcus sp. WH8109.
In line with the safety features of this laboratory, Mrs. Verdoodt also showed us the ethidium bromide workspace. As our team was performing numerous PCR gel electrophoresis experiments, the Wet-lab team needed to get accustomed to the workbench. Ethidium bromide can act as a carcinogenic substance; thus, it was of utmost importance that no object should get out of this workspace. For instance, we were not allowed to wear the same pair of gloves outside of the workspace. In brief, what stays in the ethidium bromide bench stays in there.
Although no risks nor pathogenicity was found to be associated with our cyanobacterial strains, it was essential to know that we were using imported cyanobacterial strains (both UTEX 2973 and WH8109). Therefore, there was a risk that these imported cyanobacteria can contaminate the environment that is not native to Belgian aquatic ecosystems. This risk was circumvented by only allowing the growth and manipulation of the bacteria inside the laboratory. Afterward, all the bacteria were killed with bleach following the conclusion of experiments, preventing any unwanted release into the environment. The used growth media were also properly disposed of and each compound was thrown into a corresponding waste disposal container.
In addition to using cyanobacteria, our iGEM project also involved working with cyanophages, specifically, the cyanophage S-TIP37. No threats to humans exist involving phage S-TIP37 as it only infects the freshwater strain Synechococcus sp. WH8109. Nonetheless, the prevention of cross-contamination of these phages between other stocks of cyanobacterial cultures in the laboratory was made. The propagation and manipulation of these phages were done under the laboratory of Prof. Rob Lavigne. Only a few members of the Wet-lab team could enter this laboratory, compared to the lab mentioned earlier; this was to ensure the most optimized and safe working space in Prof. Lavigne’s laboratory. Similar to the previous laboratory, safety protocols were created. For instance, the selected Wet-lab team members had also undergone a separate HSE training course before entering this laboratory, which was led by Mrs. Marleen Voet.
There has been an increasing awareness of bioethics with regard to questions and problems that are unprecedented to current times. Since the field of biology has been evolving at a fast pace, subjects such as synthetic and artificial life, animal research and GMOs, have raised several ethical questions that were not faced before . As we worked with GMOs and GMMs, our team was also interested in these ethical implications. Therefore, we consulted Prof. Pascal Borry, the program director of POC Bioethics in the Faculty of Medicine in KU Leuven, in order to give us a more in-depth view of bioethics.
The use of GMOs has been controversial due to questions regarding the impact on naturally occurring organisms. Safety then becomes a concern since there may be unforeseen circumstances that we cannot control. Common arguments for this include the potential gene transfer to other microorganisms, the growth of new microorganisms, and the proliferation of pathogenic microorganisms .
However, it should be known that genetic modifications of microorganisms can occur naturally without human intervention. With some microorganisms possessing a short generation time, mutants can be created continuously in microbial populations. Also, in vitro recombinant bacteria may lose their ability to grow or even survive in their natural habitat . However, the release of their DNA upon death and cellular lysis can still be taken by other surrounding microorganisms in the environment.
Despite the low risks that can be posed by GMOs or GMMs, there is still a possibility of other bacterial strains that can survive both in vitro and in their natural habitat. For instance, genetically improved strains of Sinorhizobium meliloti have been noted to survive in soils for six years . Thus, the complex nature of various microbial populations remains challenging to assess, resulting in uncertainty. Even though genetic modifications can occur naturally, the genetic modifications performed by researchers are not usually possible to occur in nature. For instance, the production of a thermostable alpha-amylase by Bacillus licheniformis has been genetically optimized for industrial use .
Another notable example is the engineering of Escherichia coli and Saccharomyces cerevisiae for commercially producing the human hormone, insulin . While it may sound harmless to have insulin secreted in nature, what if something more potent were to be produced an released by the Escherichia coli? That is a grave problem as there is a possibility that this E. coli can mutate into something more dangerous. Although unlikely, the possibility is still there. So, the release of GMOs into the environment becomes more of a risk with the unknown genetic modifications that can be released to nature.
Although the risks mentioned above may sound threatening, GMOs, or GMMs may be used prospectively in the production of enzymes, drugs, and other proteins. The use of GMOs or GMMs can also be economically valuable in terms of using industrial methods of production. As long as the GMO or GMM is not released into the environment, then the advantages of using them in the industry is not a complete threat when biocontainment is controllable.
One last ethical concern that was raised is the risk of overhyping a technology. More specifically, the act of overselling a product may cause misleading information to vulnerable consumers. For instance, when it became reasonably accessible to clone Escherichia coli and other microorganisms in the laboratory, it became a growing hobby as well for the public [6, 7]. Do-It-Yourself (DIY) biology is known as a biotechnological social movement that allows individuals to perform genetic engineering experiments from the comfort of their home, also known as biohacking. Though the intentions are good (for the sake of spreading biology to the public), it now becomes a threat. Unfortunately, there is a possibility that people will use this technology for the worst ideas instead of for society’s benefit. There is a large number of unknown consequences that we are not ready to face, yet when someone uses this DIY biotechnology for evil.
With the uncertainty posed by the use of GMOs or GMMs, our team has consulted the Directorate-General of Health and Food Safety of the European Commission, whereby they generously invested their time to explain to us the policies surrounding this field.
According to the Regulation (EC) No 1829/2003 on genetically modified food and feed, authorization should first be granted before an individual can place GMOs for food use, food containing or consisting of GMOs, or food produced from or containing ingredients from GMOs. In order to obtain authorization, an application must be submitted, fulfilling several criteria in which one of them includes an assessment of the potential risks for the environment and organisms. Further information on its criteria can be found in Article 5, Section 1, Chapter II . Please note that these authorizations are valid throughout the EU. After authorization, labeling is required to provide information to consumers. GM food or feed products that contain more than 0.9% in the proportion of the food or feed ingredients must be indicated with “genetically modified” or “produced from genetically modified [name of the organism]” . In line with this, no label is needed for products produced by GM organisms (e.g., T-shirt from GM cotton does not require a label). It should also be noted that these regulations also include the genetic modifications done by cyanophages, similar to our OCYANO project using marine cyanobacteria and cyanophages. However, each type of microorganism is labeled differently from each other. In the case of our project, since we plan to release completely purified enzymes to the market, labeling is not necessary as we do not intend to expose the GM cyanobacteria but only the secreted pure enzyme.
Biocontainment on an industrial scale
The risks of exposing GMOs to the outside environment have been heavily emphasized in the previous subsections, it becomes evident for our team to look at biocontainment strategies. Already discussed in the previous section under Laboratory Safety how we make sure that all biological wastes must be inactivated before environmental exposure. However, as we would like our project to have a more significant impact on society, we will be focusing on biocontainment techniques that prevent further risks of GMOs on an industrial scale.
First, as a rule of thumb, double-walled bioconversion tanks can be used to prevent the escape of our genetically modified cyanobacteria and cyanophages. As we are using genetically modified cyanophages, thorough deactivation should be applied to the remaining solution to prevent uncontrolled release to the environment and cross-contamination. There is, however, an advantage of using a specific phage strain as it can only infect a narrow set of strains. As of now, it was reported that the cyanophage S-TIP37 we used only infects our marine cyanobacterial strain (WH8109). It should also be noted that we are dealing with a diluted concentration of genetically modified cyanophages, which lowers the probability of cross-contamination. Nevertheless, precautionary measures should still be applied in the case of uncertainties.
For the decontamination procedures, chemical inactivation using chlorine (or bleach) and UV germicidal irradiation (UVGI) are the commonly employed methods . As utilizing UVGI is an energy-intensive process, whereas our group aims to promote sustainability, we plan to use chemical inactivation. Using a chemical inactivation, as low as 0.1% to 1% of bleach, can already be used in deactivating phages in a short amount of time of 30 seconds to 5 minutes . Please consider that a dechlorination step is added after this to prevent the release of chlorine that can harm aquatic life.
In line with the biosafety regarding genetically modified cyanobacteria, one recent study has demonstrated a biocontainment technique that utilizes the phosphite (Pt) dependency of Synechococcus elongatus PCC 7942 as its sole P source . By engineering Pt dehydrogenase and hypophosphite transporter genes to the cyanobacteria, this modified Synechococcus elongatus PCC 7942 can selectively assimilate Pt but not phosphate (Pi) and cannot grow on any media containing other types of P compounds. Since Pt is the nonmetabolizable form of P for most organisms and is cheap , our team can further explore this strategy in preventing contamination for large-scale operations instead of using antibiotics, which are often expensive; this strategy could be adapted to both cyanobacteria strains that we are using. As Pt is also rarely present in natural waters, the accidental release of our genetically modified cyanobacteria will not be able to survive due to this Pt-dependency. Therefore, this technique can be highly beneficial for the biocontainment of our genetically modified cyanobacteria.
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