Team:Valencia Biocampus/Safety

The organisms and parts that we require for the execution of our proyect are:

• Caenorhabditis elegans: A nematode from the Rhabditidae family, transparent and 1 mm long. It usually grows in soil and has two sexes: hermaphrodites and males. In 1963, Sydney Brenner suggested it as a great model organism due to its simplicity, easy growth, low price and the fact that it can be stored frozen for long periods of time. It is commonly used in studies such as animal development, cellular differentiation, aging and so on. Another great characteristic is the silencing method that can be implemented in this worm: interference RNA. C.elegans is included in biosafety level 1.
• Escherichia coli: It is a Gram-negative bacterium commonly found in the lower intestine of endothermic organisms. Although most strains are harmless, some can cause severe food poisoning. For our project we used E. coli strain XL1-Blue which is harmless. This strain is often used as a food source for C. elegans and is a uracil auxotroph. It is considered as Level 1 Biosafety.
• Pseudomonas putida : It is a Gram-negative bacterium. It is saprotrophic and can be found in soil. It is broadly used in bioremediation and it is completely safe. In our case we worked with P. putida KT2440 due to its ability to produce polyhydroxyalkanoates or PHAs, a biodegradable bioplastic. This strain is also a Level 1 in the WHO scale.
• Xenorhabdus nematophila and Yersenia pestis: We have NOT worked with these organisms directly but we have used one of their operons: hmsHFRS. This operon is used for the making of the biofilm as a result of its adhesion properties. Biofilms are usually considered as a virulence factor in harmful species due to its capability to attach themselves to the host organism. We have used it as part of one of our BioBricks. However, none of these genes are harmful nor represent a danger to public safety because in nature this operon is specifically design to fit C. elegans and not humans.

Once we have presented the organisms involved, we proceed to the study of its risks. We also include all the lab equipment and chemicals that could be a biological hazard:

• Organisms: Since all organisms and parts employed belong to risk group 1, we did not have to consider any special measures. However, normal lab security was always taken into account. All biological residues were correctly processed and thrown away. Autoclave and common sterilization devices were always applied to the residues. In addition, all the work places were correctly sterilized after their use, for example by UV light.
• Ethidium bromide (EtBr): It is an intercalating agent used in techniques like agarose gel electrophoresis. EtBr has health risks due to its mutagenic capacity, however in laboratories the quantity is below the level of toxicity. Even though really low levels are used, the correct handling measures were followed: double gloves wearing, lab coat, cytotoxic disposal bins, etc.
• Octanoic acid and oleic acid: At very high levels they are considered dangerous. Nevertheless, the amount with which we worked is almost impossible for it to be hazardous. All residues were correctly disposed of and then processed.

• Other chemicals: We did not use any other chemicals or products that are not used in a common molecular or microbiology laboratory. All of these possible chemicals were handled and disposed of following standard protocols: correct storage, controlled use, organized and monitored disposal containers and correct processing of the residues by the local recycling service of our university. All hazardous or dangerous work or chemical treatment was always supervised by one of our team instructors.

After analyzing all our microorganisms and products we can affirm with certainty that none of our material, work or ideas raise any safety issues towards the researchers, the public safety or the environment. All organisms and chemicals were correctly handled, as stated above, avoiding this way any danger to the researchers. Public and environmental safety were also not at risk due to the fact that all organisms are level 1 Biosafety according to the WHO and correctly managed. To conclude, even though environmental safety could seem to be affected at some point of our work, all experiments are guaranteed to follow the standard safety protocols and organisms are never released into the environment. In addition, all microorganisms are correctly taken care of at all times.

None of the BioBricks or vectors used in our constructs present a potential risk to human health. Our work was developed with strains of P. putrida and E.coli (both of them are included in a biosafety level 1) which are easily killed by autoclaving or using bleach.

One of our BioBricks contained an operon (hmsHFRS) that comes from Xhenorhabdus nematophilus, a nematode-dependent parasite. That operon is also found in Yersinia pestis (cause of the Black Death); an organism qualified with a BSL 2 or 3 (depends on the strain). However, we did not work with the organisms described above and the concrete operon (that allows the formation of a biofilm on Caenorhabditis elegans) has no effect in humans.

Because of this, no special measures have been required either in the registry or laboratory work.

In our laboratory of Biotechnology and Synthetic Biology at Cavanilles Institute (Valencia) there is not a biosafety group. However there is a biosecurity committee from the University of Valencia that has recently visited the laboratory where the team has been working in order to confirm that all the safety rules are accomplished. We are currently working at a P1 laboratory and next year’s target is to transform it in a P2.

Manel Porcar, our main supervisor, is in the national committee of biosafety. In our institute, all the actuation elements to prevent accidents such as fire, explosions or poisoning are available in every laboratory room. Emergency exits, eyebaths, first-aid kits, extinguishers and special waste containers can be found in all the building.

To face possible safety problems that could appear in future iGEM competitions, we have thought that improving the communication between teams should be a first step.

Probably, it would be a good idea to create a social platform joined by both instructors and students that could allow the direct communication between all the participants that have taken part through the years in the iGEM. But we consider even more important an indirect and clear form of communication, that is, it would be nice to possess a database where we could find how to work with the different organisms that have been used as chassis (protocols, managing and important points to deal with when working with them).

In the same way, it would be nice to promote a major detail in the description of the different BioBricks: detailed conditions in which they have been used and the ways they have respond after the treatment, organisms in which they have been tested and other considerations. All this would make the scientific search of information easier, because it could be arranged, for example, by type of cells.

The aim of increasing communication deals, for example, with the problem of the security of the BioBricks sent by the teams. When registering BioBricks, their safety suggestions are included, but indeed nobody checks this. What we propose is that all teams could check the security of the BioBricks they want, that have been sent to the registry before, and as a reward they would obtain extra points. Firstly, in silico toxicology as non-testing methods could be done in order to obtain information about the structure of the protein or the gene sequence, and once with these early results, they could be compared with analogous structures or sequences that are dangerous for humans. After that, also in vivo test could be done using animals like mice in toxicity testing methods. This could be divided in two parts: providing them with high doses of the BioBrick product (high toxicity) and low doses in long-term periods (chronic toxicity). This last part would only be done by teams that had the right lab structures and the necessary security systems.

In conclusion, with these experiments we would have more information on whether the BioBricks are safe for humans or not. Finally a ‘Safety Committee’ could check previous studies in order to minimize the potential risks and ensure that studies are properly made.