Team:UNITS Trieste/Safety

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 lab?
b.   risks to the safety and health of the general public if released by design or accident?
c.   risks to environmental quality if released by design or accident?
d.   risks to security through malicious misuse by individuals, groups or states?

Specifically, are any parts or devices in your project associated with (or known to cause):
-   pathogenicity, infectivity, or toxicity?
-   threats to environmental quality?
-   security concerns?

We made several meetings to make a risk evaluation based on the Italian and European Community guidelines and finally concluded that neither the SynBiome nor the new Biobricks the team wants to create represent a risk to safety and health as specified in the points a, b, c, d. We considered different aspects in our analysis.

In the bacterial part of the SynBiome Project we intend to use a strain of Escherichia coli, that is categorized as Risk Group 1 microorganism, while the cells we use for the eukaryotic section are Hela cells. Considering that the laboratories we work in are both BSL 2 (Biosafety Level 2), according to the Italian laws, the danger is absent or low for workers and community.

We also considered the risks related to the vectors used, the donor and the receiving microorganisms, the Biobricks used and the genes transferred and transfected.
Our bacterial constructs contain promoters and genes that produce molecules for Quorum Sensing communication, its regulators (derived from Agrobacterium tumefaciens and Pseudomonas aeruginosa) and a β-glucosidase by Cellulomonas fimi.
We didn’t use C. fimi and P. aeruginosa because the parts we needed were already available in the Registry. We had to extract gDNA for new Biobricks from the A. tumefaciens bacteria, which are safe for human health, but pathogenic for plants. However our project didn’t foresee the genetic manipulation of these species and we managed them very carefully during the extraction procedures, in order to avoid their spreading in the environment. The TRA-nslator and LAS-“n”lator constructs are introduced in a pBBR1MCS-3 expression vector, that derived by pBBR1MCS cloning vector (see Kovach et Al., 1995 for more information). The constructs containing the glucosidase are cloned inside a standard plasmid of the Registry.
All these constructs mentioned are introduced in an E. coli DH5α strain and the resulting organisms are still Risk Group 1 bacteria. Moreover, the system is designed so that the QS molecules and the corresponding regulator are produced by two different modified DH5α strain. In this way, they aren’t dangerous for the environment because they need each other to produce the proteins we introduced.
We used modified strains of Pseudomonas putida and A. tumefaciens as biosensors to quantify the lactones produced by our constructs. P. putida requires the same attention of A. tumefaciens.

The eukaryotic cells are modified in order to constitutively produce a transcriptional regulator and an inducible β-lactamase. We used two different commercial plasmids for these constructs, pCDNA3 and pIRES2-EGFP that are well characterized by the producing companies. The antibiotic resistance is produced only in the presence of certain QS molecules produced by bacteria (see Neddermann et Al., 2003). This is necessary for the bacterial survival in the Synthetic Biome. Anyway, in the absence of the genetically modified bacteria, these Hela cells won’t be able to transcribe for the enzyme, so it doesn’t represent a sensitive risk. Plus, if accidentally spread outside the lab, the Hela cells won’t be able to maintain the DNA transfected.

Under what biosafety provisions will / do you operate?
a.   Does your institution have its own biosafety rules and if so what are they?
Provide a link to them online if possible.

The ICGEB (International Centre for Genetic Engineering and Biotechnology) has a Biosafety Unit that is especially focused on the effect of the new technologies on the environment. To learn more about the ICGEB goals in this field, click here).
The ICGEB centre of Trieste, where the team works, doesn’t have its own rules, because it respects the Italian laws (see the following points).

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

We don’t have an Institutional Biosafety Committee or equivalent group. The legal responsible about safety in the laboratories the team is working in, is the ICGEB Director Dr. Mauro Giacca, who is following the development of the project since the beginning. The institute has a Safety Assistant as well, Dr. Marco Vegliach, who has been aware about SynBiome. When the team’s project was explained, they didn’t find any particular danger concerning the safety and security beyond the usual, so no change was needed.

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

We are aware that the manipulation of bacteria and eukaryotic cells has always a range of risk for human and environmental safety. So before startingthe activity of the wetlab, the team asked to the Safety Assistant about the Italian laws and rules followed by the ICGEB for its laboratories. He gave us a copy of the WHO’s Laboratory Biosafety Manual, explaining us what it means to work in a BSL 2 laboratory.

On the other hand, we have been trained by the responsible of each laboratory about different aspects of the Biosafety and Security at work place. The key points are:
   -   the general behavior rules we must follow, like wearing gloves and lab coat when we manage chemicals and cellular cultures, never eating or drinking inside the work area and smoking outside the building;
   -   the equipment we need to use, as the UV transilluminator, the laminar flow cabinets, the biosafety hoods, the confocal microscope and the autoclave for tubes and glassware used for cellular cultures and bacteria;
   -   the containment and the waste handling procedures, in order to avoid the involuntary spreading of microorganisms.
However during the wetlab practice, we were steadily aware by our instructors about how to behave when managing bacteria and some chemicals.

d.   Does your country have national biosafety regulations or guidelines?
If so, provide a link to them online if possible.

The laboratories that accommodate our team follow the Dlgs 81/08 about health and safety issues at workplace, the Dlgs 206/01 and the DM 25.09.2001 (Italian texts only) about the restrained use of genetically modified microorganisms, as suggested by the 2000/608/EC, the 98/81/EC and the 90/219/EEC directives.

Do you have any other ideas how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

We think that for our SynBiome the safety rules suggested by the iGem competition can be considered as adeguate.