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Welcome to iGEM Panama Wiki

Our SynBio Project for iGEM 2011

There is considerable interest among the bio-industries in pharmacology and bioremediation products such as rhamnolipids. Rhamnolipids have proven healing and diseases fighting properties in curing or ameliorating psoriasis, decubitus ulcers, burns, and wounds, and there are indications that rhamnolipids will aid in the treatment of a range of dermatoses or exzemas. Since rhamnolipids can be used to form liposome, there are potential applications for using rhamnolipids for drug delivery. The use of rhamnolipids as biosurfactants is important in the remediation of oil spill areas. The cleanup of the Exxon Valdez oil spill with rhamnolipids as biosurfactants was too expensive and complicated, therefore impractical for large-scale bioremediation. However, advances in genetic engineering as is synthetic biology can make rhamnolipids a viable solution to oil spill pollution cleanup, enhanced oil recovery, sludge removal, pesticide dispersal and wetting agents. In this project we modify a naturally occurring gene which synthesizes rhamnolipids to make it compatible with Assembly Standard 10 for rhamnosiltransferase 1 complex (rhlAB) gene expression in Escherichia coli for standardized rhamnolipid production. We submitted the first rhamnosyltransferase BioBrick to the Registry of Standard Biological Parts and this achievement earned us a bronze medal at the IGEM (International genetically Engineered Machine)2010 competition. This is the second time a Panamanian team participated in this Synthetic Biology contest and we became pioneers in Panama for trying to develop the Synthetic Biology field, a promising new applied science. In this second step of our project, (1) we will integrate other genetic standarized parts plus our rhlAB_BB to build a expression platform to create an E. coli-based factory, improving the efficiency and optimization of rhamnolipid biosynthesis using the BioBrick standard. (2)We will be focus in chemical, biological and physical characterization of our rhamnosyltranserase BioBrickTM (rh1AB_BB). Some version of these devices may even include hydrocarbon sensors for Just-In-Time production of rhamnolipids.

Contents 1 SAFETY - How to establish criteria for risk? 1.1 Risk Criteria for Establishing Ascending Levels of Containment [1] 2 Pseudomonas aeruginosa 2.1 Pathophysiology[2] 2.1.1 Biosafety Level 2 [1] 3 A. Standard Microbiological Practices 4 B. Special Practices 5 C. Safety Equipment (Primary Barriers and Personal Protective Equipment) 6 D. Laboratory Facilities (Secondary Barriers) 7 References

SAFETY - How to establish criteria for risk?

Risk Criteria for Establishing Ascending Levels of Containment [1]

(Extract from Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition.)

The primary risk criteria used to define the four ascending levels of containment, referred to as biosafety levels 1 through 4, are infectivity, severity of disease, transmissibility, and the nature of the work being conducted. Another important risk factor for agents that cause moderate to severe disease is the origin of the agent, whether indigenous or exotic. Each level of containment describes the microbiological practices, safety equipment and facility safeguards for the corresponding level of risk associated with handling a particular agent. The basic practices and equipment are appropriate for protocols common to most research and clinical laboratories. The facility safeguards help protect non-laboratory occupants of the building and the public health and environment. Biosafety level 1 (BSL-1) is the basic level of protection and is appropriate for agents that are not known to cause disease in normal, healthy humans. Biosafety level 2 (BSL-2) is appropriate for handling moderate-risk agents that cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. Biosafety level 3 (BSL-3) is appropriate for agents with a known potential for aerosol transmission, for agents that may cause serious and potentially lethal infections and that are indigenous or exotic in origin. Exotic agents that pose a high individual risk of life-threatening disease by infectious aerosols and for which no treatment is available are restricted to high containment laboratories that meet biosafety level 4 (BSL-4) standards

Pseudomonas aeruginosa

We must remember that the purpose behind our project is to take a gene that produces the Rhamnosyltransferase 1 enzyme of a pathogenic bacteria known as Pseudomonas aeruginosa and insert it into an efficient and not pathogenic strain of Escherichia coli. This will help biosynthezise a rhamnolipid that can aid in the more efficent degradation of hydrocarbons. For this reason, we must take into consideration that once the gene Rh1AB is inside the e.coli is not longer toxic. Another important point to mention is that Pseudomonas aeruginosa does not appear in HHS AND USDA Select Agents and Toxins list. The question that arise is (mainly for not bilogist): Is the P. aeruginosa a pathogenic organism?, What are the measures to manipulate and work with P. Aeruginosa safely?

Pathophysiology[2]

Pseudomonas aeruginosa is an opportunistic pathogen. It rarely causes disease in healthy persons. In most cases of infection, the integrity of a physical barrier to infection (eg, skin, mucous membrane) is lost or an underlying immune deficiency (eg, neutropenia, immunosuppression) is present. Adding to its pathogenicity, this bacterium has minimal nutritional requirements and can tolerate a wide variety of physical conditions.

The pathogenesis of pseudomonal infections is multifactorial and complex. Pseudomonas species are both invasive and toxigenic. The 3 stages, according to Pollack (2000), are

1. bacterial attachment and colonization,
2. local infection, and
3. bloodstream dissemination and systemic disease. The importance of colonization and adherence is most evident when studied in the context of respiratory tract infection in patients with cystic fibrosis and in those that complicate mechanical ventilation. Production of extracellular proteases adds to the organism's virulence by assisting in bacterial adherence and invasion.

Table 1: Resistance of selected organisms to decontamination is presented in descending order. If dangerous and highly infectious agents are present in a laboratory, the methods for decontamination of spills, laboratory equipment, biological safety cabinets (BSC), or infectious waste are very significant and may include prolonged autoclave cycles, or gaseous treatment of surfaces.[1]

Biosafety Level 2 [1]

Pseudomonas aeruginosa requires Biosafety level 2, appropriate for handling moderate-risk agents that cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure[1].

The work was done under strict biosafety Level 2 as required by this bacteria, so it does not represent any risk to the researchers health.

Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents that pose moderate hazards to personnel and the environment. It differs from BSL-1 in that: 1) laboratory personnel have specific training in handling pathogenic agents and are supervised by scientists competent in handling infectious agents and associated procedures; 2) access to the laboratory is restricted when work is being conducted; and 3) all procedures in which infectious aerosols or splashes may be created are conducted in BSCs or other physical containment equipment. The following standard and special practices, safety equipment, and facility requirements apply to BSL-2.

We must note that we do not use for our BioBrick the whole P. aeruginosa organism. We performed a genomic DNA extraction and isolate our gene of interest. From this point there is no danger in handling. For bioremediation applications, the idea is not release the bacteria into the environment but release the produced rhamnolipid. For this reason does not represent any risk to public health or for the ecosystem. However, thinking about a scenario in which accidentally we released the bacteria containing the gene Rh1AB, will not be a serious safety problem . This is because the gene Rh1AB per se is not toxic, and within our BioBrick expression is absolutely secure.

A. Standard Microbiological Practices

(Extract from Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition.)

1. The laboratory supervisor must enforce the institutional policies that control access to the laboratory.
2. Persons must wash their hands after working with potentially hazardous materials and before leaving the laboratory.
3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.
5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include:
   • Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
   • Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal.
   • Non-disposable sharps must be placed in a hard walled container for transport to a processing area for decontamination, preferably by autoclaving.
   • Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible.
6. Perform all procedures to minimize the creation of splashes and/or aerosols.
7. Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant.
8. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method. Depending on where the decontamination will be performed, the following methods should be used prior to transport:
   • Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak proof container and secured for transport.
   • Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
9. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present. Posted information must include: the laboratory’s biosafety level, the supervisor’s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the laboratory. Agent information should be posted in accordance with the institutional policy.
10. An effective integrated pest management program is required.
11. The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions toºprevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of childbearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance.

B. Special Practices

(Extract from Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition.)

1. All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements.
2. Laboratory personnel must be provided medical surveillance, as appropriate, and offered available immunizations for agents handled or potentially present in the laboratory.
3. Each institution should consider the need for collection and storage of serum samples from at-risk personnel.
4. A laboratory-specific biosafety manual must be prepared and adopted as policy. The biosafety manual must be available and accessible.
5. The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-2 agents.
6. Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility.
7. Laboratory equipment should be routinely decontaminated, as well as, after spills, splashes, or other potential contamination.
   • Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
   • Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.
8. Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures described in the laboratory biosafety manual. All such incidents must be reported to the laboratory supervisor. Medical evaluation, surveillance, and treatment should be provided and appropriate records maintained.
9. Animal and plants not associated with the work being performed must not be permitted in the laboratory.
10. All procedures involving the manipulation of infectious materials that may generate an aerosol should be conducted within a BSC or other physical containment devices.

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

(Extract from Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition.)

1. Properly maintained BSCs, other appropriate personal protective equipment, or other physical containment devices must be used whenever:
   • Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include pipetting, centrifuging, grinding, blending, shaking, mixing, sonicating, opening containers of infectious materials, inoculating animals intranasally, and harvesting infected tissues from animals or eggs.
   • High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory using sealed rotor heads or centrifuge safety cups.
2. Protective laboratory coats, gowns, smocks, or uniforms designated for laboratory use must be worn while working with hazardous materials. Remove protective clothing before leaving for non-laboratory areas, e.g., cafeteria, library, and administrative offices). Dispose of protective clothing appropriately, or deposit it for laundering by the institution. It is recommended that laboratory clothing not be taken home.
3. Eye and face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays of infectious or other hazardous materials when the microorganisms must be handled outside the BSC or containment device. Eye and face protection must be disposed of with other contaminated laboratory waste or decontaminated before reuse. Persons who wear contact lenses in laboratories should also wear eye protection.
4. Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Gloves must not be worn outside the laboratory. In addition, BSL-2 laboratory workers should:
   • Change gloves when contaminated, glove integrity is compromised, or when otherwise necessary.
   • Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
   • Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. Hand washing protocols must be rigorously followed.
5. Eye, face and respiratory protection should be used in rooms containing infected animals as determined by the risk assessment.

D. Laboratory Facilities (Secondary Barriers)

(Extract from Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition.)

1. Laboratory doors should be self-closing and have locks in accordance with the institutional policies.
2. Laboratories must have a sink for hand washing. The sink may be manually, hands-free, or automatically operated. It should be located near the exit door.
3. The laboratory should be designed so that it can be easily cleaned and decontaminated. Carpets and rugs in laboratories are not permitted.
4. Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
   • Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
   • Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant.
5. Laboratory windows that open to the exterior are not recommended. However, if a laboratory does have windows that open to the exterior, they must be fitted with screens.
6. BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, windows that can be opened, heavily traveled laboratory areas, and other possible airflow disruptions.
7. Vacuum lines should be protected with liquid disinfectant traps.
8. An eyewash station must be readily available.
9. There are no specific requirements for ventilation systems. However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without recirculation to spaces outside of the laboratory.
10. HEPA filtered exhaust air from a Class II BSC can be safely recirculation back into the laboratory environment if the cabinet is tested and certified at least annually and operated according to manufacturer’s recommendations. BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or directly exhausted to the outside through a hard connection. Provisions to assure proper safety cabinet performance and air system operation must be verified.
11. A method for decontaminating all laboratory wastes should be available in the facility (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method).

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