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Biosafety

Safety questions?

1. Would any of your project ideas raise safety issues in terms of:

    1.	researcher safety,
    2.	public safety, or
    3.	Environmental safety?

A= The answer for this question, is based principally, in the fact that we are working in a Basic-Biosafety level 2 Laboratory (INDICASAT-AIP), with a DNA modified Escherichia coli bacterium, and a native Pseudomonas aeruginosa bacterium, corresponding to the Risk Group 2 (moderate individual risk, low community risk); a pathogen that can cause human or animal disease but is unlikely to be a serious hazard to laboratory workers, the community, livestock or the environment. Laboratory exposures may cause serious infection, but effective treatment and preventive measures are available and the risk of spread of infection is limited.

Like we mention above, that kind of microorganism does not represent a serious hazard to laboratory workers, the community, livestock or the environment, but is important to understand that we should have care when we are using that kind of microorganisms because laboratory exposures may cause serious infections; basically digestive system´s infections and urogenital system´s infections.

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,

    1.	did you document these issues in the Registry?
    2.	how did you manage to handle the safety issue?
    3.	How could other teams learn from your experience?

A= This year 2011, we are trying to get a new Biobrick of Rhamnose, that is a six carbons monosaccharide, found on bacteria natural habitats, like a component for the biosynthesis of Rhamnolipid. The DNA sequence for the biosynthesis of Rhamnose is a natural structure in Pseudomonas aeruginosa´s genome. We´ll obtain this DNA sequence from an Environmental P. aeruginosa strain, and finally We´ll put this sequence on an expression platform. Now, we know that Rhamnose is an organic component of all kind of ecosystem, for this reason rhamnose does not represent a hazard for the human, animal or environment life.

3. Is there a local biosafety group, committee, or review board at your institution?

    1.	If yes, what does your local biosafety group think about your project?
    2.	If no, which specific biosafety rules or guidelines do you have to consider 
       in your country?

A= In our institution don´t exist any committee or biosafety group, but we have some rules or guidelines for work with biological hazards, we learned on INDICASAT-AIP Laboratories some kind of general lab procedures (standard operating procedures SOPs) like : • Autoclave all the instruments that have contact with biologic compounds before discard. • Put all the trash that had contact with biologic compounds in a proper bag (designed red bags for hazardous materials). • Use biological safety cabinets (with HEPA filters) when you are using a hazard material. Clean the cabinet before and after you use it. • Discard the cutting material on adequate recipients (Plastic recipients), before and after autoclave. • Use a lab coat always! And gloves. • Clean everything (sterilize) with alcohol (70%) and Chlorine solution (15%).

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

A= For future iGEM competitions will be really useful, in the deal with safety issues, the use of prepared kits by the same competition(of course according with the standards protocols you are using), not commercial kits! We talk about kits developed by iGEM directions. Such as: Miniprep Kits, Purification and Mutagenesis Kits, etc. that ensures a low environmental risk, an excellent discard of hazard materials such as liquids and aerosols, decreasing simultaneously the health risk of all the students that are involved in iGEM´s Competitions.

Additional Considerations

In our SynBio Project for iGEM 2011, we are using Recombinant DNA Technology trying to create a microorganism capable of degrade hydrocarbons (or similar substances). Taking a DNA sequence from Pseudomonas aeruginosa and inserting that sequence in an Escherichia coli strain (K-12); our superpowerful bacteria will be capable of synthesize the Rhamnosyltransferase 1 enzyme.

This enzyme is able to biosynthesize with simple nutritional complements, structures called β-hydroxyalkanoic acids, which are only the lipid part of a rhamnolipid. The 2010’s Panama iGEM Project was created with the same idea of the 2011´s Panama iGEM project, but this year we are trying to characterize our biobrick, and create a bacterium, that with simple media can create a complete rhamnolipid (including the rhamnose part of the rhamnolipid-absent in the 2010´s Panama iGEM project).

Is really important understand, that recombinant DNA technology involves combining genetic material from different sources thereby creating genetically modified organisms (GMOs) that may have never existed in nature before, (World Health Organization, 2004).

For this reason is necessary to establish some biosafety standards, and some others considerations when we are working with our modified Escherichia coli; beginning with the description of our two involved bacteria.

Escherichia coli K-12 is a non-pathogenic strain that cannot permanently colonize the gut of healthy humans or animals, then, routine genetic engineering experiments can safely be performed in E. coli K12 at Biosafety Level 1, provided the inserted foreign DNA expression products do not require higher biosafety levels.

Higher biosafety levels may be required when: 1. The expression of DNA sequences derived from pathogenic organisms may increase the virulence of the GMO. 2. Inserted DNA sequences are not well characterized 3. Gene products have potential pharmacological activity. 4. Gene products code for toxins.

In our project with E. coli K-12 strain, the rh1 AB DNA sequence, just have the nucleotides needed for create β-hydroxyalkanoic acids, and is a completely safety insert that does not increase the virulence of the GMO. Besides, the rh1 AB was completely characterized (not in E. coli; but in other microorganisms has been characterized) and hasn´t gene products code for toxins.

Our biobrick has potential pharmacological activity, for this reason we are working in a Biosafety level 2, and we are not thinking in liberate this GMO to the environment (maybe in the future…).

We said healthy humans or animals, because inmunocompromised individuals are susceptible of infection, including when are affected by the Pseudomonas aeruginosa native´s strain.

Some hazards that we should consider from the donor organism (who has the DNA sequence of interest-in our case Pseudomonas aeruginosa) are:

1. Pseudomonas aeruginosa has multiple antibiotic´s resistance. 2. Pseudomonas aeruginosa has three antigen classes O (Somatic-specific for this specie), H (Flagellate) and M (mycoides). 3. P. aeruginosa has different components and everyone has different virulent properties: • Adhesin: Union´s mechanism for join to Host cells • Exotoxin A: Inhibition of the protein synthesis on the hepatic cells, heart, kidney, lungs and spleen. Inhibition of amino acids catchment at cellular level. • Cytotoxin: This substance can affect the majority of the Eukaryotic cells, incrementing the membranes’ destabilization and decreasing the bactericidal effects. • Exotoxin B: This substance can increment the bacterial pathogenicity. • Enzymes:

 Elastase: This substance can affect the arterial walls cells.  Collagenase: It can produce cornea´s infection.

This bacterium has three different levels of infection which are: 1. Union and bacterial colonization. This bacterium can join to the host cell through Pili or Fimbriae to the respiratory epithelium, bucal cells, damaged trachea, and tracheobronchial mucin. 2. Local Invasion: through enzymes. 3. Dissemination and systemic Invasion: through toxins. Some pathologies associated with Pseudomonas aeruginosa are: native and prosthetics valve endocarditis, hospital-acquired pneumonia, meningitis, neutropenic and burned septicemia.

References

[1]National Institutes of Health 2009 Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition, U.S. Department of Health and Human Services, Public Health Service Centers for Disease Control and Prevention; , HHS Publication No. (CDC) 21-1112.

[2]Qarah, S., Cunha B., Dua,P., Lessnau, K. 2009 Pseudomonas aeruginosa Infections eMedicine. URL:http://emedicine.medscape.com/article/226748-overview