1. Would the materials used in your project and/or your final product pose:

a. Risks to the safety and health of team members or others in the

The biological device in which we worked on this project doesn’t pose a risk to any of the team members’ health if correctly handled, however we should always consider that they carry an antibiotic resistance. Nevertheless, we must also take in mind the chemicals employed in the methods used for the completion of this project. In order to dispose any of those hazardous substances, besides following the General Safety Regulations of the Laboratory, we also followed the Safety Regulations of Centro de Biotecnología FEMSA. For example, in agarose gel electrophoresis ethidium bromide and SYBR green are commonly used; in this case, we followed a protocol for handling and disposal of the chemicals, placing objects contaminated with it in special containers. In the case of pipette tips, those were treated with benzal and sterilized by autoclave. These measures were done to avoid direct contact between team members and hazardous material.

b. Risks to the safety and health of the general public if released by design or accident?

Concerning the risks to people in general, the most dangerous problem we observed is the exposure to microorganisms that are resistant to antibiotics, posing a threat on the treatment of many diseases. For this reason, we concealed our bacteria for lab use only following the appropriate measures for handling and disposing them, such as sterilizing the tubes and dishes containing modified bacteria after using them. Regarding chemical substances, we assured that every chemical used was properly disposed, even gloves used by the team when they made contact with any hazardous substance, to make sure no people alien to the lab could be exposed to the chemicals.

c. Risks to environmental quality if released by design or accident?

Our biological devices pose few risks to the environment since we are using enzymes already present in other bacteria and non-dangerous substrates and products. However, there is a slight environmental concern in one of our constructs. This construct employs cellulose as a substrate, and since all plants have cellulose in their cell wall it is possible that they could be degraded under unusual circumstances, thus generating a negative environmental impact. Again, caution measures must be taken in order to have the modified bacteria concealed so they don’t pose a direct threat to the environment.

d. Risks to security through malicious misuse by individuals, groups or states?

Our project doesn’t have any point of risk in regards of biosecurity. Due to the nature of the process and the microorganisms involved, the devices do not cause any hazard that could be misused by individuals, groups or states to affect people’s security and wellbeing.

Please explain your responses (whether yes or no) to these questions. Specifically, are any parts or devices in your project associated (or known to cause):

- pathogenicity, infectivity, or toxicity? Neither of our devices has an associated risk in these fields. The only possible concern could be the EstA protein used in the device, because it is capable of the production of a toxin when the entire sequence is used; but since only a partial sequence of this protein is needed in the device (only the membrane expressing part), the toxin cannot be synthesized and the risk is therefore eliminated.

- threats to environmental quality? There is a very little, if none at all, concern about threats on the environment by our devices. As previously explained, if modified bacteria with the cellulase gene are misused or escape, there is a possibility that plants will have contact with them, creating a possible environmental damage especially if the affected organisms are ecologically valuable. However, this scenario is more a concern than a menace, as bacteria need to grow under specific conditions, and plants themselves have defense mechanisms against pathogenic microorganisms, so that possibility is greatly diminished.

- security concerns?

No, our project does not posess any danger to security in general, because the enzymes are designed only to convert sucrose into glucose and fructose, and cellulose on glucose. Regarding the rest of the devices, the parts only complement the enzymes to allow them to express and function properly, so they don’t pose a security threat.

2. If your response to any of the questions above is yes:

a. Explain how you addressed these issues in project design and while conducting laboratory work.

When designing our project, thanks to the opinion of many professors, we considered the risks that our device could have because of the enzymes and the reaction catalyzed. Interestingly enough, the enzymes are already used in other industries such as food and cleaning industries, so the measures taken to confine the bacteria were effective in case there was a risk to the environment or health in general. To accomplish an acceptable safety level, during the work time at the laboratory, every time bacteria with the constructs were handled, said work was done inside a laminar flow hood, disinfected before and after being used. Disposed materials were placed in red bags for biological hazardous waste, and then sterilized in autoclave. In the end, the material was stored in red containers so they could be disposed by a certified company.

3. Under what biosafety provisions will / do you operate?

a. Does your institution have its own biosafety rules and if so what are they?

Up to this date, there is not a general biosafety handbook for the university’s laboratories. Nevertheless, we used the handbook provided to us by the university’s research center, that consists in a handbook for handling and disposal of hazardous biological waste and a handbook with the safety rules that must be followed within the lab (link) . Thanks to this, we were able to take the appropriate safety measures in the academic lab where we worked along the summer for this project.

b. Does your institution have an Institutional Biosafety Committee equivalent group? If yes, have you discussed your project with them?
Describe any concerns or changes that were made based on this review.

Yes, the university’s research center, “Centro de Biotecnologia FEMSA”, has a Biosecurity Committee, composed of three doctors from the institution. All of them received information about our project, the laboratory protocols that we were using, and the biosafety measures we had; this was done to have our project checked by them so it could be improved. The Commitee told us that everything was in order and that we were following appropriately the measures taken in the university. However, one of them noted a point that we had overlooked: that the use of cellulase in a construct could affect the environment because cellulose is the main component of plants, representing thus a little biosafety concern. For this reason, the Commitee suggested us to follow the already established biosafety regulations, to not accidentally free transformed bacteria into the environment.

c. Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.

Before initiating the project, some of our team members had already been to a conference about the laboratory rules that must be followed in the university’s Research Center, “Centro de Biotecnologia FEMSA”. The rest of us went to this conference when the project started. In this conference, the Research Center coordinators provided us with the rules that we had to follow in those laboratories. The rulebook was adapted to the academic laboratory where we worked, the Genetic Manipulation and Molecular Diagnosis Laboratory (link). Also, Marco Mata, head of the Research Center Safety Commission, supported us in the biosafety aspect of the project, informing us about the biosafety measures followed in the Research Center, and allowing us to take the same measures to our lab to work along the summer.

d. Does your country have national biosafety regulations or guidelines?

In Mexico, in matters of biosafety, there is a Regulation for the handling of Hazardous Biological Waste, which we followed to properly dispose all the waste generated by the project. (link). Moreover, there is a Biosafety Law for Genetically Modified Organisms (link).