Team:Brasil-USP/Notebook/Safety
Collaborations
Brasil-USP team is very concerned about the project’s biosafety, and considered it not only while working in the wet lab but also while designing our biological system. Below you can find more information about how our team handled this subject.
Safety Training
All of the students involved in our project have received safety instructions for working at the laboratory. Graduated students have attended a course offered by São Carlos Institute of Physics (IFSC/USP) about basic safety aspects, and then instructed undergraduate students about the precautions to be taken. The course included topics such as: the use of appropriated clothing and personal protective equipment; correct use of equipments such as autoclave, laminar hood flow, electrophoresis chamber and centrifuge; handling safety aspects and disposal of biological and chemical agents; specific danger of the main reagents used at the lab; and precautions about bioaerosol. All of the knowledge obtained in the course was applied on the project developed for iGEM.
Risks of The Project
Parts
The goal of our iGEM project 2015 is to produce roxA (Rubber oxygenase) and Lcp (Latex clearing protein) in E.coli under laboratory or industrial conditions. Both of these proteins are not entirely elucidated, but it is known that they are responsible to degrade poly(isoprene) into smaller molecules. The main molecule produced in this process is ODTD (12-oxo-4,8-dimethyltrideca-4,8-diene-1-al), however there are no reports about the threat that this compound offers to the environment and to humans. Both of proteins used only degradates the polymer of the rubber, therefore, they don’t offer any harm to humans once we do not have this polymer constituting cell process. Our circuit uses inducible promoters (BBa_K91403; BBa_R0080; BBa_R0010), so if the trigger reactant isn’t present our genic circuit is off, not producing the ODTD molecule, which might be toxic to the environment.
Chassis
According to the DSMZ database, Acidithiobacillus ferroxidans and Acidithiobacillus thiooxidans are from the risk group 1, and does not offer any disease on healthy adult humans[i]. Additionally, the E. coli BL21, contains several mutations silenced, and does not compete in the environment. There have been no reports showing serious harm in humans. Therefore, this strain is classificated in Risk group 1, and it meets the requirements of the US NIH[ii]. For the Interlab study, we used E. Coli DH5-α. Both of them are also from risk group 1, presenting no serious threats.
Precautions taken to avoid unexpected release of the GMO
Bioreactors
While scaling up our project, bioreactors will be used for rubber devulcanization and degradation of natural rubber. These acts as a physical containment for the developed bacteria and, therefore, minimize the risks of releasing it unexpectedly to the environment3.Also, as these processes are being idealized to be performed in factories, only few people of the staff would have contact with the developed microorganism; and these would be specifically trained to minimize contamination - granting an even lower chance of the microorganism being released unexpectedly. Unfortunately, physical containment alone isn’t enough to ensure that the GMO’s won’t be released accidentally. Physical containment is normally enough to ensure that GMO’s won’t be released accidentally, however it’s not 100% efficient. Therefore, other precautions were also taken, as described below.
Kill Switch
A kill switch mechanism designed to be activated when the bacteria leaves the bioreactor was included in our DNA circuit. For this purpose, our team has worked with a simplified Hok-Sok mechanism - a highly studied kill switch mechanism that is effective on most gram-negative bacteria, and even in some gram-positive microorganisms4. This is how it works: the bacterial culture medium will contain rhamnose, activating the left part of our designed DNA circuit (Figure 1). This will lead to the production of tetR, which inhibits the formation of hokD. If the bacteria is released from the bioreactor, it will be in an environment without rhamnose, and won't produce tetR - leading to the production of HokD, a protein that causes cell membrane depolarization, killing the microorganism4. The mechanism is expected to kill the cells almost immediately, with only a fraction of 1,4 x 10-4 the cells surviving after 90 minutes4.Therefore, the system is efficient for containing the microorganism spreading and its modified DNA transmission.
Figure 1. DNA circuit proposed by our team.
Although the simplified Hok-Sok kill switch mechanism proposed by our team allows, with low energy cost, the efficient and rapid death of the cells after leaving the bioreactor, it doesn’t grant that cells inside the bioreactor which didn’t inherit the developed DNA circuit die. This may be a problem to the process efficiency, as cells that aren’t developing the desired processes will be competing for nutrients with the ones that contain the DNA circuit. For the proposed project, these contaminations will be controlled using antibiotics. As the bacteria desired to survive will contain a plasmid with antibiotic resistance, it won’t die - and all of the other bacteria will. As the continuum use of antibiotics can lead to resistant bacteria and has been avoided by scientific community5, our team proposes a circuit that would be able to control these problems. It will not be constructed due to time issues, but it is idealized here (Figure 2).
