Team:USP UNIFESP-Brazil/Safety

AlgAranha Team USP_UNIFESP-Brazil

iGEM 2016

Our project design: risks that may arise and possible measures

In this project, we intend to explore the modular characteristic of spider’s silk proteins, through synthetic biology techniques and generate a modular bio nanomaterial able to immobilize proteins. This bio-nanomaterial will be composed of modular recombinant proteins from spider’s silk, which will be the immobilization support to other proteins with antimicrobial properties (enzybiotic endolysins). By using these properties, the ultimate focus will be the use of this technology to develop artificial skin for burn injured people.

It is important to note that our ultimate aim with this project would be to applicate the wound dressing made of spider’s silk on the burn wound.In theory, would speed up the healing process, decreasing the length of stay in hospital, very common in burned victims. In this way, our customized biomaterial product could be applied on the skin of burn victims. Thus, our product would be used by patients with infection, helping to heal infections (caused by resistant bacteria) after burn accidents. The use of our product however, will be restrict to the hospital.

Project development: laboratory level

Genetically modified organisms (GMOs) are organisms “whose genetic material has been artificially modified to change their characteristics in some way. To develop our project we used organisms listed in the table 1 as host for our genetic constructions or as models to test our product. The organisms are classified in biosafety levels 1 and 2, according to the criteria of Brazilian National Technical Commision on Biosafety (CTNBio). Organisms classified in biosafety level 1 are non pathogenic, for animals and humans, however, when an organism is classified for the level 2, the risk of infection exists, despite being minimum (BRASIL. Instrução Normativa nº 7, de 06 de junho de 1997).

Table 1: Organisms we planned to useused in our project

Organism	Biosafety Level	Strains used
Escherichia coli	1	DH10B, DH5a, JM110
Chlamidomonas reinhardtii	1	cc1690
Staphylococcus aureus (not used)	2	ATCC 25923

Escherichia coli is a well established organism commonly used in research laboratories all over the world. This gram-negative bacterium presents a rapid growth rate, simple nutritional requirements, well-established genetics and genomic sequence. Staphylococcus aureus (ATCC 25923) is a level 2 organism and will be used in an enzyme activity test. Basically, this strain will be exposed to different concentrations of our enzybiotics, in order to test their effectiveness, in a proper location, according to the rules for biosafety level 2 laboratories. Finally, Chlamydomonas reinhardtii is a microalgae and a model organism in genetic recombination with high content of CG in its genome, a favorable feature to the expression of spider silk proteins.

Some concerns may raise when research is developed using GMOs, such as genetic interbreeding, competition with natural species, horizontal transfer of recombinant genes to other microorganisms, adverse effects on the health of people or the environment, ethical issues and unpredictable and unintended effects (reference). All these issues (and others that may raise) can be addressed at different levels: from laws and guidelines to molecular mechanisms of containment to human behaviour and social context.

By law (law number 11.105/2005), in Brazil, all institutions that use genetic engineering methods must create a Internal Biosafety Committe (CIBio) and name a researcher for each project developed. All internal committees of each institution that works with GMOs within USP respond directly to the CTNBio, created in 1995, by the Biosafety Law (federal law Nº 8974, of January 5, 1995). All CIBios are responsible for monitoring laboratories where experiments are developed with genetically modified organisms and also report any accident to the CTNBio. Thus, their main function is to monitor all the stages associated to GMO and its derivatives’ manipulation and to ensure that the laws and guidelines are properly followed.

At the Faculty of Pharmaceutical Sciences of the University of São Paulo (FCF-USP) laboratory that has being used for our experiments, the chief researcher is doctor professor João Carlos Monteiro de Carvalho and the CIBio chairman is doctor professor Mario Hiroyuki Hirata.

It is important to notes that the context about where the research is being developed is “essential in identifying circumstances that create, amplify, or diminish risk” [2]. The unique features of synthetic biology allied with the fact that this field, and our project included, can be carry out of the labs of the University, was seen by us as a matter of safety concern.

Wolfe et al., (2016) show in their paper that “much of the risk related synthetic biology literature references social and institutional elements such as biosafety committees and formal rules or guidance, but typically not in terms of how those elements translate into practice”. With this quote in mind, we decided that every member of our team should know the basics concept of biosafety and biosecurity. Thinking about that, we held a meeting to discuss and learn about the laboratory practices and techniques. This meeting was headed by our instructor, PhD student João Dutra Molino, and occurred in the same laboratory where our experiments have being done. Then, team members could learn where to find every equipments and how to manipulate them properly. Moreover, other team members also develop research projects in laboratories from different institutes at our university (undergraduate and postgraduate research projects). All these students received guidelines about safety in their respectives laboratories. Thus, during our training, we helped each other and also discussed about safety. The focus in our training was to learn how to use individual protection equipment, how to deal with our host organisms in a safe way and how to use reagents in a proper manner, being focused in the manufacturer's specifications.

Molecular mechanisms

In 1975, at the Asilomar conference, it was stablished stringent guidelines for DNA manipulation and research (Berg, 2008). It has incorporated physical and biological containment into experiments to reduce environmental risks by preventing the recombinant/synthetic DNA or engineered organism spread .

Some molecular mechanisms can be adopted to develop safer organisms and it can be incorporated in our project, producing organisms that are not efficient in transfer ou spread our engineered genes, mainly the genes associated to bacteria resistance. For example, dependence devices can be construct or order to avoid GMO survival outside a controlled environment, such as a laboratory. These devices can use a auxotrophy system (when the organism does not have an essential gene, such as a gene for an essential amino acid. This gene is expressed, instead, from a plasmid), or a system that allows plasmid expression (containing the recombinant\synthetic DNA) or protein translation just in organism that was engineered. Basically, these devices are constructed to make plasmid propagation dependent on a specific host or to keep the organism viability dependent on maintaining a plasmid (Wright, Stan and Ellis, 2013) Different devices has been developed and a discussion about the advantages and pitfalls of some of them can be found somewhere else.

Product commercialization and final user concerns: drug resistance

Despite of the potential benefits of our product, drug resistance issues could also arise. Besides the concerns about how to treat infections caused by resistant bacteria, another concern is related to the improper antibiotic use.

The modern use of antibiotics started in 1928 with discovery of penicillin. The microbial infections are a old practice in human history and were management in Ancient world, especially Egypt, China and Greece. The use of antibiotics changed the medicine through 2 effects: “reducing mortality and increasing life expectancy” (Blair et al, 2015). Regardless this improve, a large number of diseases are caused by multidrug-resistant bacteria, due the improper antibiotics use and this number is increasing in a global context (Ventola, 2015).

Bacteria resistance can be a result of a resistance that was acquired or as something already present in the organism. These “intrinsic mechanisms” embrace apparatus that improve the resistance for some specific antibiotic as a consequence of “inherent structural or functional characteristics”. The bacterias can act decreasing the intracellular concentrations of the drug due a poor efflux or penetration inside the bacteria or even modifying the target of the antibiotic by post-translational or genetic modifications (Blair et al, 2015).

One important concern with this matter is the fact that several bacterial pathogens associated with human diseases have evolved into MDR - multidrug-resistant- forms after the antibiotic use (Davies and Davies, 2010). In this sense, the development of measures to avoid this type of behavior by health professionals and society in general must be done before our product (or any product with the same features) commercialization. Improvement of diagnostic and prescribing practices in the hospitals, optimization of treatment regimens, avoiding long-term patient exposition to antibiotics and preventive campaigns, in order to avoid bacterial infections are measures that can be adopted to minimize bacteria resistance spread (Ventola, 2015).

Finally, our modular silk could be used as an immobilization support not just for endolysins, but also for other enzymes in different industrial processes. So, our product would be used in different kinds of factories, as a consumer product that ordinary people buy and to be used directly in the human body. All these points have to be considered individually and specifically for each application, to assess the possible risks related to our final product and its production process. </p>

REFERENCES

Berg, P. (2008) Meetings that changed the world: Asilomar 1975: DNA modification secured. Nature. 455:290-291.

Blair, J. M. A., et al (2015) Molecular mechanisms of antibiotic resistance. Nature Reviews Microbiology. 13: 42-51.

Davies J, Davies D. (2010) Origins and Evolution of Antibiotic Resistance.Microbiology and Molecular Biology Reviews : MMBR. 74(3):417-433. doi:10.1128/MMBR.00016-10.

Ventola CL. (2015) The Antibiotic Resistance Crisis: Part 1: Causes and Threats.Pharmacy and Therapeutics. 40(4):277-283.

Ventola CL. (2015) The Antibiotic Resistance Crisis: Part 2: Management Strategies and New Agents. Pharmacy and Therapeutics.40(5):344-352.

Wright, O., G-B. Stan, and T. Ellis. (2013) Building-in biosafety for synthetic biology. Microbiol. Papers in Press. Microbiol. 159: 1221-1235.

Wolfe, A.K. et al., (2016). Synthetic Biology R&D Risks: Social–Institutional Contexts Matter! Trens in Biotechology. 34(5):353-356.