Team:Waterloo

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Revision as of 22:49, 27 October 2010

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Staphiscope

Abstract

Superbugs, or antibiotic resistant microorganisms, are microbes that have become resistant to traditional treatments. These types of infections are difficult to diagnose, treat, and eradicate, making the healing process time consuming and resource intensive. The native quorum-sensing unit from S.aureus (the Agr system), will be introduced into a non-pathogenic strain of E.coli. The E.coli will then effectively have the ability to eavesdrop on the activity of the pathogenic organism and emit an indication of the magnitude of the infection in the form of RFP. Using sensitivity tuners the system can be designed such that the response will occur at an exact level, when the size of the population poses a threat to the host. Upon a positive result from a diagnosis, further tests could be done to specify whether MRSA (methicillin-resistant S. aureus) or MSSA (Methicillin-sensitive S. aureus) are present.



Modelling

Since the project aims to detect S. aureus at low concentrations, the modelling aims to determine how sensitive the system will be so that it can be adjusted to detect S. aureus at clinically-relevant concentrations. To that end, models for the sensitivity tuners from the 2009 Cambridge team needed to be built that can be combined with models for the AIP detection system. Most importantly, the noisiness of the system needs to be determined to know if false positives will be generated.


Sponsers

Department of Biology NEB Canada WEEF NSERC SFF
Project
Modeling
Outreach
Human
Practices

1.0 INTRODUCTION


Methicilin resistant Staphylococcus aureus (MRSA) are bacteria whose presence has been quite problematic in terms of human pathogenic infections. Since its discovery in the 1880s, Staphylococcus aureus has been the known cause for several kinds of minor skin, and major post-surgical infections. Prior to 1940, mortality rates in relation to this pathogenic organism reached 80% (Deurenberg and Stobberingh, 2008.) The first wave of resistant S. aureus came two years after the introduction of penicillin for medicinal practice in 1940. In less than twenty years, most of the S. aureus strains known to man were unaffected by the antibiotic. In the late 1950s, a penicillinase-resistant penicillin was brought forth to the medicinal market; this was methicilin (Deurenberg and Stobberingh, 2008.) The bacterium's subsequent resistance to methicilin came two years after its introduction in 1959. The mecA gene responsible for methicillin resistance is also to blame for the organisms’ desensitization to several classes of antibiotics (Deurenberg and Stobberingh, 2008). This has led to the creation of the term: MRSA, now commonly used worldwide.


While an MRSA infection is much like a S. aureus infection, the difference comes in the lack of sensitivity of the MRSA to several classes of antibiotics. This makes MRSA infections a serious threat in both health and economical aspects.


The infection could be easily transferred directly (through regular skin-to-skin contact), as well as indirectly (through contamination of surfaces). The ease with which infection could occur, as well as the high mortality rate involved with the infection, make MRSA a serious threat to the health of patients worldwide (Durai et al., 2010.)

Apart from health issues, the high treatment costs associated with MRSA infections are also of priority. The treatment of MRSA infected patients may result in intravenous antibiotics being taken, as well as a prolonged hospital stay. A recent study found that the costs of such a treatment can range between $8 364 and $13 940 per patient. This means that one patient’s treatment is approximately a fourth of the cost of an annual MRSA screening for the staff of an entire hospital. As the spending associated with treatment will far out-weight that of screening, it is very important that efficient and low-cost screening methods be implemented in hospitals (Durai et al., 2010.)


There are only a few methods, which currently exit for MRSA screening. These are plating, liquid-broth inoculation, and PCR (polymerase chain reaction) assays. While these will be discussed in further detail later on in the report, it is important to note that these methods could be costly and time consuming, and could sometimes present incorrect results (Durai et al., 2010.)


The International Genetically Engineered team at the university of Waterloo has been working on a method which will be used in supplement to the already-existing ones for the detection of MRSA infections. It is a proof of concept for diagnosing the presence of Staphylococcus aureus in an infection site before it has had the chance to create an infection. This synthetic biology–based diagnostic method will take advantage of the quorum sensing mechanism of Staphylococcus aureus, and utilize Escherichia coli as the sensor and reporter


2.0 CURRENT METHODS USED FOR THE DETECTION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS


Early detection and treatment of MRSA carriers, and persons with developed infections is of grave importance. A simplified grouping of the methods available to hospital staff for diagnosis of MRSA presence is presented below.


2.1 CONVENTIONAL METHODS


These methods have been around for a long time and involve basic plating and inoculation assays. They are quite labor and resources intensive. This is especially true nowadays, as a greater amount of tests needs to be completed to accommodate the increased number of potential patients involved with MRSA, either as passive carriers or as infection victims (Fang and Hedin, 2003.)


2.1.1 PLATING METHOD

This method usually involves two series of platings. The first one is inoculation onto one or both of Mannitol Salt Agar plates, and Blood Agar plates. The inoculated solid media then need to be incubated at 35°C for 24 - 48hrs and 18 - 24hrs respectively (Warren at al, 2004.) After growth is observed, one may identify S. aureus as a yellow colony on the mannitol plate, and as a cleared out zone on the Blood Agar, further confirming with Gram staining. Specific tests can be completed by streaking the resulting single colonies onto antibiotic plates for MRSA identification. This, however, takes up another 18-24hrs at the least. This method is very time consuming and potentially inappropriate for the time-dependent nature of infections. It is also highly resource intensive in its requirement for the technicians’ time, as well as the lab’s plates, agar, antibiotics, etc. Nonetheless, it presents 92.8% specificity in differentiation between various S. aureus, and 98.5% sensitivity in recognizing positive versus negative results (Warren at al, 2004.)


2.1.2 ENRICHMENT BROTH METHOD

This particular method utilizes liquid broth, which is to support the growth of MRSA or S. aureus in general. Depending on which of the above is chosen, inoculation of the original sample could occur into one of the following broths – MRSA: Iso-Sensitest broth 2.3% NaCl, 1 g of aztreonam/ml, and 2 g of oxacillin/ml (Fang and Hedin, 2003); or S. aureus: tryptic soy broth and 6.5% sodium chloride (Warren et al., 2004). These are left overnight, and are to be re-plated on the next day, on the same types of pates as described in section 2.1 above. Unfortunately, the enrichment step proves unworthy, as sensitivity is decreased to 92.4% and specificity, to 91.4% (Warren et al., 2004.) This method also proves very tedious, time and resource consuming.


2.2 RT-PCR METHODS


The above-mentioned methods are considered to be, although fairly reliable, extremely slow. Plating and sensitivity testing are time-consuming processes, which put the previously-mentioned techniques at a minimum delay of 20 to 24 hrs (Warren et al., 2004.) The quickest method available for the detection of Methicillin-resistant Staphylococcus aureus is a PCR assay. Unfortunately, PCR assays are very expensive as compared to other techniques. The cost of some PCR assays (including reagents and technicians’ time) may be approximately $9 CAD per test if done in-house, and around $40 CAD if not (Mahony et al., 2004.) PCR involves the use of primers, which will recognize a sequence particular for MRSA and amplify it, producing a visible fluorescence –detectable result during the amplification stage (Elsayed et al., 2003.) While various testing facilities may use different genes limited to S. aureus, the most commonly used sequences for MRSA detection through PCR are the mecA (responsible for methiciilin resistance) and nuc (S. aureus species-specific marker) genes (Elsayed et al., 2003.) The two methods described below reveal differentiating ways of

1.preparing the samples obtained from patients to be analyzed, and
2.the PCR techniques which follow in accordance with the sample preparation.


2.2.1 BROTH-PCR METHOD

This method involves the use of enrichment broth, which will show preference towards nuc-carriers with mecA in the same cell (Milsson, Alexandersson and Ripa, 2005.) Samples from patients would be taken and inoculated into the above-mentioned enrichment broth and incubated overnight. Results could be obtained on the next day using PCR. The nuc-specific primers which have been used in previous trials with the method are (Fang and Hedin, 2003):

a. NUC1 (5-GCG ATT GAT GGT GAT ACG GTT-3)

  • b. NUC2 (5-AGC CAA GCC TTG ACG AAC TAA AGC-3)

  • The broth PCR technique has been tested to give 93.3% sensitivity, 89.6% specificity, 31.8% positive predictive value, and 99.6% negative predictive value. As the percentage reliability of negative results is very high, especially as compared to that of the positive predictive value, the application of the broth-PCR method will be able to report the negative samples on the morning after testing. This will, in the long run, save as much as 84.9% of the cost and labor which is associated with the necessity of further testing (Fang and Hedin, 2003).


    2.2.2 IDI-MRSA DETECTION METHOD

    The IDI (Infectio Diagnostic Inc) nasal swab method provides an assay, which completely removes the need for inoculation in liquid or solid media. The assay allows for the performance of PCR directly from a nasal swab sample, using reagents and methods provided by the manufacturer (Infectio Diagnostic Inc). While sample preparation differentiates, PCR methodology applied is quite similar to the one described in section 2.2.1 above. There are, however, distinct differences between the results produced by the two methods. For one, the IDI-MRSA detection method can be complete in as little as 1.5hrs post sample collection, versus next day with the above-mentioned method, and at least 24hrs with the plating methods. As well, The IDI-MRSA provides 91.7% sensitivity, 93.5% specificity, 82.5% positive predictive value, and 97.1% negative predictive value when compared to culture-based methods (Warren et al, 2004.)


    3.0 STAPHISCOPE – A SYNTHETIC BIOLOGY PERSPECTIVE ON MRSA DETECTION


    As could be noted, there are not many methods available for the detecting of this pathogenic organism. StaphiScope, iGEM Waterloo’s 2010 project, has been designed as a supplement to the methods described above. This diagnostic tool has the goal of sensing and reporting for the presence of S. aureus populations in an area of interest (e.g., a wound) prior to virulence. It will do so faster than any of the described plating methods, and at a lower price. The method relies very heavily on the quorum sensing capabilities of Staphylococcus aureus.


    3.1 QUORUM SENSING: WHAT IS IT AND WHY IS IT SO IMPORTANT?


    Quorum sensing is defined as “the communication mechanism that enables bacteria to make collective decisions” (Boyen et al., 2009.) This mechanism involves the release of signaling molecules to the cell’s exterior. In gram-positive bacteria such as S. aureus, these signaling molecules are referred to as auto-inducing peptides (AIPs). The signaling molecules could then be detected by special receptors on other bacteria (not necessarily of the same strain) and give feedback in regards to the population density at the site. Bacteria rely on quorum sensing in order to thrive as a population. Most importantly, however, at certain critical concentrations of the AIPs, the bacterial populations make a collective decision to, for example, initiate virulence (Boyen et al., 2009.)


    This year, the UW iGEM team is attempting to listen in on the quorum sensing “conversations” of S. aureus, by engineering E. coli to receive the AIP signaling molecule mentioned above. Through a signal amplifier, E. coli would be able to detect the presence of S. aureus at very low concentrations, and prior to the bacteria having the chance to become virulent.


    3.2 PRACTICAL APPLICATION OF STAPHISCOPE


    The purpose of StaphiScope is to provide the option of speeding up some of the methods mentioned above, without compromising their reliability. It will be a particularly useful tool in speeding up the conventional methods of serial plating. In particular, the plating method described above requires that samples first be plated onto either Mannitol Salt Agar in order to obtain yellow colonies, or Blood Agar to obtain zones of clearance. This will aid in picking out Staphilococcus aureus apart from other species which may have grown. Only after this initial growth can we plate on antibiotic plates containing β-lactam class of antibiotics (methicilin, penicillin and a few others). This would normally take up another, at the very least, 18-24 hours. Using StaphiScope, we could speed up the first step of this process, by picking out the Staphilococcus aureus from all other species without the requirement of growing them up on solid media overnight first. With StaphiScope, the bacterial population from the samples would be grown into liquid culture, and the presence of RFP will be evaluated. The following will be involved in using the StaphiScope method commercially:

    Obtain sample from patient on cotton swab. This will, for example, be from a wound.
    Obtain liquid LB culture and inoculate into it a colony (from a plate containing many) of StaphiScope E. coli. These are E. coli species containing BioBrick K359009 – final construct. These plates could be kept in stock for quick access.
    Inoculate sample from patient into liquid media containing StaphiScope and place at 37°C for 3 – 4 hrs. In our experience this is sufficient to show color of RFP is it has been activated. In addition, it would be useful to spin the cells down through a centrifuge (2 min at 13, 000 rpm). This would bring the cells to the bottom, allowing us to see the red color expressed more clearly.
    Upon positive result (red color produced), the cells could now undergo either plating on media containing the methicillin class of antibiotics, or a PCR-detection method using nuc/mecA genes (as described in section 2.2.1)


    This method would bring the time consumption demands down to a few hours, as opposed to at the very least, a day. As well, less media would be used in the process. Finally, it is much cheaper than the faster methods such as PCR.


    It is possible to initially inoculate the sample from the patient into liquid broth containing samples of the methicillin class of antibiotics. The expression of RFP in this case would mean that MRSA are present. This direct method of detection could shorten the test to 3-4hrs in length. However, this may lead to false positive results, as it is possible for other bacteria to release AIP as well, activating the StaphiScope.


    3.3 TECHNICALITY OF THE STAPHISCOPE S. AUREUS DETECTION TOOL


    The primary design of the construct consisted of AIP sensor infrastructure BBa_I746101 + BBa_I746104, permeability device BBa_I746201, amplifier, and reporter. Amplifier would have been selected from BBa_K2743xx series based on the investigations by the modeling team. Red fluorescent protein gene BBa_E1010 from BioBrick I13507 will be used as reporter. Please refer to Figure 1 below for more information.


    Figure 1: Primary design of StaphiScope project, incorporating sensor, amplifier and reporter of AIP.


    An additional design involved the addition of a generator part. This would have allowed E.coli to produce its own AIP, as a means for testing the response without worrying about permeability into the periplasm. This design was completed but could not be tested due to time constrains. As well, no amplifier was added in the final construct. Below is a discussion of the major components of StaphiScope.


    3.3.1 COMPONENT #1: FEP A PERMEABILITY DEVICE

    As S. aureus releases AIP in order to initiate any kind of quorum sensing, it is important that Escherichia coli will be able to receive and properly identify this signal. As E. coli is a gram-negative bacterium (contains an additional outer membrane), an issue arises of AIP not being able to get through this extra layer. This is why the iGEM team was forced to use FepA as part of the plasmid construct. FepA is an outer membrane permeability device, which will allow for AIP (the quorum sensing signaling molecule which will come from S. aureus) to get through the outer membrane of the gram-negative E. coli. The permeability device comes with its own promoter, on which we will rely for optimal expression.


    3.3.2 COMPONENT #2: AIP SENSOR

    It is important that AIP does indeed make it past the outer membrane of E. coli, as this is crucial to turning on the sensor and reporter functions of the StaphiScope. The AIP sensor function will allow E. coli to “listen in” on the quorum sensing conversations of the S. aureus. The BioBrick is modeled after the agr (accessory gene regulator) quorum sensing system present in S. aureus. AIP (from same cell or from another cell producing the peptide) binds AgrC (a histidine kinase). This, in turn, allows for phosphotransfer to AgrA and subsequently, through activation of transcription, more AIP is produced, amplifying the signal. It is a loop (Atkinson and Williams, 2009.) This is part of the mechanism which allows the bacteria to be aware of the concentration of its own strain in the infected area, subsequently leading to a “collective decision” such as expressing virulence. Please refer to Figure 2 for a visual display of the previously mentioned.


    Figure 2: agr quorum sensing system in S. aureus (Cambridge iGEM, 2008)


    3.3.3 COMPONENT #3: SIGNAL AMPLIFIER

    This BioBrick will serve the purpose of further amplifying the AIP signal obtained by E. coli, and thus, allowing it to “sense” S. aureus, before S. aureus has had the chance to produce a high enough concentration of the quorum sensing signal to initiate a response. In other words, E.coli would be able to, through amplification, “sense” S. aureus before S. aureus has had the chance to sense itself. Unfortunately, there was insufficient time for proper research to be done on this, and so, the final construct lacks an amplifier. The amplifier is not a necessary part, but simply an assurance that the signal will be received through over-amplification.


    3.3.4 COMPONENT #4: RFP REPORTER

    This RFP BioBrick will produce a red-fluorescence signal proportional to the AIP concentration E. coli senses. This way, not only would one be able to tell that S. aureus is present, they would also be able to quantitatively compare samples to see where the infection has spread further. This would mean being able to decide whom to treat first, in the worst case scenario.


    3.3.5 COMPONENT #5: AIP GENERATOR; PERIPLASM TEST.

    The purpose of this construct will be to check the sensor-reporter system. The alternative construct contains AIP Generator for self-induction and no permeability device to facilitate accumulation of AIP in the periplasm. The system would essentially product its own AIP, thus showing activity of sensor-reporter system before final tests in presence of S. aureus.


    3.4 BIOBRICKS SUBMITTED


    As a supplement to the following information, please refer to the construction tree presented in Figure 3 below.


    Figure 3: Construction tree of StaphiScope



    K359003 - This part, which has been submitted to the registry, contains a P2 promoter and an RFP reporter.
    K359006 - This part, which has not been submitted to the registry, contains the AIP sender/generator and sensor/receiver parts.
    K359007 - This part, which has been submitted to the registry, contains the FepA permeability device into the sensor/receiver construct.
    K359008 - This part, submitted to the registry, is the testing construct. It contains the sender/generator, sensor/receiver and reporter parts.
    K359009 - This is the final construct of StaphiScope. It contains the permeability device, as well as the sensor/receiver, and reporter parts.

    3.5 THE QUANTIFICATION INITIATIVE


    Characterization of certain parts, functioning as signal amplifiers, was done by the iGEM Team Cambridge, which designed them, in terms of arabinose as an input signal. The arabinose sensing system consists of promoter pBAD and it respective repressor AraC. The BioBrick part BBa_I0500 contains both of those components in one part, while BBa_I13453 and BBa_I13458 are similar to BBa_I0500 split into two components: pBAD and AraC respectively. In order to retrieve the data about Team Cambridge’s parts, the modeling division of iGEM team Waterloo 2010 requested characterization of pBAD in relative promoter units (RPUs). The article by Kelly et al. (2009, Measuring the activity of BioBrick promoters using in vivo reference standard) describes the issue of great deviations involved with measuring biological systems due to too many factors to account for. The proposed solution is to measure in reference to a standard: the promoter BBa_J23101. The proposed vector for both the measured part and the standard is pSB3K3 (low copy number kanamycin resistant BioBrick plasmid) with green fluorescent protein gene as reporter. Inspired by swappable promoter construct BBa_J61002 similar construct with J23101 in place of swappable promoter element was derived from pSB3K3, the BBa_K359201.


    Figure 4


    RFP (BBa_E1010) will be used instead of GFP due to consistent failure to express GFP (BBa_E0040) reported by Waterloo iGEM teams from previous years. The fluorimetric assay can be performed on a fluorescent protein with the use of flow cytometer or microplate reader. The two approaches focus on different aspects of the retrievable data. Flow cytometry acquires data on individual cells, however each new concentration of input and time point requires individual assay. Incubating fluorescence-capable plate reader allows to acquire many time and concentration points overnight or even several nights, and is thus preferable. Checking back on the QC sequencing data for I0500 it was discovered that the integrity of the part in the distribution available was not succesfully confirmed by sequencing, which explains failure of RFP expression with this part. The alternative approach of assembling complete the pBAD sequence in two halves to the forward and reverse PCR primers used to amplify the vector, such that when the vector is religated the correct promoter sequence is formed. The AraC repressor can be provided on another plasmid.

    4.0 FUTURE PLANS


    The AIP molecule utilized by S. aureus as part of its quorum sensing mechanism is part of a group of 4 classes of signaling molecules, all with the ability to cross inhibit each other’s activity. It could be speculated that, if present at the same time, they would slowly inhibit the signal transmission between members of the same infection site, and thus prevent infection. Thus, it would be recommended that, after the construction of the StaphiScope, an extension be added to the project, where within Escherichia coli is integrated the function to produce a signal molecule of the cross-inhibitory group (Atkinson and Williams, 2009)


    5.0 REFERENCES



    Atkinson, S. and Williams, P. (2009). Quorum Sensing and Social Networking in the Microbial World. Journal of the Royal Society Interface. 6, 959-978.

    Boyen et al. (2009.) Quorum sensing in veterinary pathogens: Mechanisms, clinical importance and future perspectives. Journal of Veterinary Microbiology, 135, 187-195.

    Deurenberg, R.H. & Stobberingh, E.E. (2008). The evolution of Staphylococcus aureus. Infection, Genetics and Evolution, 8 (6), 747-763.

    Durai, R., Ng, P., and Hoque, H. (2010). Methicillin-Resistant Staphylococcus aureus: An Update. Journal of Association of periOperative Registered Nurses. 91 (5). 599-609.

    Elsayed et al., (2003) Development and Validation of a Molecular Beacon Probe–Based Real-Time Polymerase Chain Reaction Assay for Rapid Detection of Methicillin Resistance in Staphylococcus aureus. Arch Pathol Lab Med, 127, 845-849.

    Fang, H. and Hedin, G. (2003). Rapid Screening and Identification of Methicillin-resistant Staphylococcus aureus from Clinical Samples by Selective-Broth and Real-Time PCR Assay. Journal of Clinical Microbiology, 41 (7), 2894-2899.

    Mahony et al. (2004). Performance and Cost Evaluation of One Commercial and Six In-House Conventional and Real-Time Reverse Transcription-PCR Assays for Detection of Severe Acute Respiratory Syndrome Coronavirus. Journal of Clinical Microbiology, 42 (4), 1471-1476

    Milsson, P., Alexandersson, H., and Ripa, T. (2005). Use of broth enrichment and real-time PCR to exclude the presence of methicillin-resistant Staphylococcus aureus in clinical samples: a sensitive screening approach. Journal of Clinical Microbiology and Infection, 11 (12), 1027-1034.

    University of Cambridge iGEM Webpage. (2007). Modeling Bacterial Quorum Sensing. Retrieved September 1, 2010, from, https://2008.igem.org/Team:Cambridge/Modelling

    University of Waterloo iGEM Team Webpage. (2010). BactoHouse: Abstract. Retrieved September 1, 2010, from http://igem.uwaterloo.ca/BactoHouse.

    Warren et al. (2004). Detection of Methicillin-Resistant Staphylococcus aureus Directly from Nasal Swab Specimens by a Real-Time PCR Assay. Journal of Clinical Microbiology, 42 (12). 5578-5581.

    1.1 Outreach and Synthetic Biology

    The newness of synthetic biology means that much of the population is not even aware that it exists (see Awareness & Attitudes Study see link at the bottom of this section). Therefore, an important aspect of our outreach efforts is to introduce the topic of synthetic biology and show its potential. We also hope to give people the foundational information that they need in order to understand future scientific developments. This form of outreach will help to improve the scientific literacy of the general population.

    Another emerging issue is misconceptions held by the general public. In the development of synthetic biology, as with many new technologies, there is still much to learn and discover. As a result, the information made available to the public is often not a comprehensive, accurate picture of synthetic biology.

    The following are the goals that we hope to achieve through educational outreach:

    • Inform the public about synthetic biology
    • Promote an education in science
    • Showcase opportunities in the field of science
    • Create an enriched science experience for students
    • Broaden the influence of iGEM

    For more information about outreach and synthetic biology see:

    by Peter D. Hart Research Associates, Inc. on awareness and attitudes of the public about synthetic biology found that 9 of 10 individuals think the public should know more about developing technologies aims to provide a complete picture on the development of new sciences examines the development of synthetic biology

    1.2 The Events

    The method that we have chosen to achieve our goals is science education. This is the avenue most accessible and familiar to us as science students. By participating in events in our community we are able to influence multiple audiences by different means. Our outreach efforts are primarily focused at young students still deciding whether to continue in the field of science, students pursing science education, and the general public. Some of the events facilitated delivering the information on a small scale (in depth discussions with one or two students) while other events necessitated speaking more generally to larger groups. The events fostered synthetic biology awareness and were great learning experiences for our team.

    ESQ Partnership

    ESQ ( Engineering Science Quest ) is a day camp hosted at the University of Waterloo that brings hundreds of curious young minds to Waterloo each year to learn more about science and engineering. We held two different weekly activities for campers of ages 8-9 and 12-14.

    The activity for children of 8 to 9 years of age involved extracting their own DNA from their cheek cells. It was approximately an hour and a half in length, and involved not only following the outlined procedure, but also, a discussion of synthetic biology, DNA, proteins, enzymes and more. At the end of each activity, the children were allowed to keep a sealed cryovial with their extracted DNA.

    The protocol for the activity, as well as the PowerPoint presentation and script associated with it, are available here.

    The second activity, which involved children 12 to 14 years of age was named “Do We Really Need to Wash Our Hands.” In this activity, the children were asked to swab their own hands (using a sterilized cotton swab) and plate the resulting swab on solid media. They would then wash their hands/use hand sanitizer and swab and plate again. The plates would be left to incubate at 37°C overnight, and the next day, the resulting growth would be presented to the children. As well, they were allowed to swab two other area of their choice, as to see how “dirty” these are. Most chose their own hair, backpack or shoe.

    Prior to each activity, a PowerPoint presentation discussion bacteria, pathogenicity, hand-washing, DNA and synthetic biology was given to the students. As well, they were provided with a hand-out to aid them in their understanding of the activity. The handout contained step-by-step activity protocol, as well as spaces for hypothesizing and discussion. The PowerPoint presentation, protocol, script, and handout are available here.

    We hope that these initiatives will help engage students outside of the classroom and get them excited about science.

    iGEM Ontario (OGEM) Meeting, June 15 2010 at McMaster University

    The second annual OGEM Meeting was held at McMaster University in Hamilton on June 15th, 2010. Members of iGEM teams from University of Waterloo, University of Western Ontario and University of Toronto gathered to discuss the future of a regional synthetic biology community, as well as a regional conference. The day turned over to discussions which centered around creating more communication and support between teams. In addition, the gathering was also an opportunity for teams to get to know one another before heading down to MIT. The meeting was a great success and the second of more regional gatherings to come.

    As the meeting was held during the annual CSM (Canadian Society for Microbiologists) conference, members were able to attend a series of lectures given by valuable members of the field. As well, iGEM Ontario members were given the opportunity to speak about their projects and promote the idea of iGEM during a poster presentation held along with researchers in the field of microbiology.

    In the future we hope to see this organization (independent of an individual iGEM team) become an important resource to Ontario iGEM teams and for educating the general public about synthetic biology. We are currently working on an iGEM Ontario website.

    Building Life: The Science of Synthetic Biology

    On June 23, Waterloo iGEM adviser, Dr. Trevor Charles, held an open public lecture aiming at discussing synthetic biology, its means and aims. During the lecture, the purpose of synthetic biology, its ethical and safety implications well as many other current topics were discussed. At the end, a facilitated panel discussion featuring Andre Masella (Waterloo iGEM team), Dr. Kathryn Plaisance (bio-ethicist), as well as Dr. Maria Trainer (Council of Canadian Academics) answered some of the public’s pending questions regarding synthetic biology. The lecture was a great success, with a large number of attendees of various scientific background. It was part of a series of lectures organized by the University of Waterloo’s Department of Biology, in attempt to increase public awareness of current scientific issues.

    What We Accomplished

    Upon reflection we feel that we have achieved the following goals through our outreach efforts:

    • Introduced synthetic biology to individuals who knew nothing about it
    • Educated multiple audiences on the fundamentals of science such that they will be able to better understand future scientific information and developments
    • Excited young minds about science
    • Strengthened our regional synthetic biology community
    • Communicated the work of other iGEM teams and the benefit and goals of the iGEM competition
    • Increased communication between local synthetic biologists
    • Gained insight and experience into what the public believes about modern science
    • Developed activities and displays to be used and improved upon in the future

    Future Plans

    We plan to continue our outreach efforts in the future. We plan to expand our efforts and continue to educate the public about synthetic biology and share our love of science. In the future our efforts will continue to center around science education. Our profile in our community is increasing as iGEM becomes a familiar group and synthetic biology ceases to sound frightening and gains familiarity. We hope that the Ontario community of iGEM teams will continue to grow and include teams from all across Canada, helping to strengthen the synthetic biology community across the country. As members of the Waterloo iGEM team we are proud to educate multiple audiences and to share our knowledge and passion with the world.

    SAFETY:

    Our project is not expected to raise any safety issues in terms of researcher, public or environmental safety. The health risks associated with the organisms used and the work completed could be classified as very low to moderate. None of the biobricks we submitted raise any safety concerns. In terms of biosafety rules to be followed, we would consider Biosafety Level 2 to be a sufficient indicator of safety requirements. This stems from the fact that the organisms involved are Escherichia coli and Staphilococcus aureus. However, in practical use (e.g., hospital setting), level 3 facilities would be required. This is to account for several factors, including the increased potential for highly pathogenic strains in the vicinity, as well as the very unlikely possibility of a pathogenic strain of Escherichia coli obtaining the AIP sensing characteristics of our StaphiScope model. In terms of biosafety issues which could be useful for future iGEM competitions, the appearance of a desired biobrick into a pathogenic strain of bacteria would be the biggest concern. The potential for such an occurrence would be limited by Biosafety Level 2 rules, as described below.

    Facilities, equipment, and procedures which are required to contain risk group 2 organisms at Level 2 have been met:

    1. Laboratory separated from other activities
    2. Biohazard sign
    3. Room surfaces impervious and readily cleanable.
    4. Equipment includes an autoclave
    5. There is personal protective equipment which includes laboratory coats and gloves worn only in the laboratory.

    All precautions with respect to recombinant DNA were observed:

    • All waste was autoclaved before being thrown away.
    • Researchers practiced aseptic technique, and frequent hand washing.
    • Bench surfaces were disinfected with ethanol.

    INTRODUCTION


    As synthetic biology expands, as pressing world problems emerge and grow, the potential impact of synthetic biology becomes more evident. Although a primary goal of developers of synthetic biology should be to consider the ethical, societal, safety, environmental, and political impact of the science, we believe that interest should also be paid unto the impact that synthetic biology will have in the business world.

    As a group we are most interested in the course of development of synthetic biology in industry; the goal of our project is to try and decipher the path that synthetic biology will forge as it expands in the business world. Our attempt to answer this question has begun with a comprehensive inquiry into important factors affecting the diffusion of synthetic biology. It is our opinion that before we attempt to make any conclusive statements about the future direction of synthetic biology or the economy as a whole we must have a complete picture of the current landscape of synthetic biology and the markets it could potentially impact.

    This inquiry is intended for use by multiple audiences; particularly scientists and members of business in relevant industries. As scientists it is important to understand the context in which discoveries are made, understanding where world needs lie and how discoveries will impact the world makes for better-informed scientists. Members of the business world should also strive to have an understanding of where the need and rationale for discoveries come from. Although profit is the ultimate endgame a well-informed approach helps to prevent ethical pitfalls and often greater success.

    This analysis looks at both extrinsic and intrinsic factors relating to the development of synthetic biology as a whole. In particular intrinsic factors such as the development of synthetic biology in specific industries (biofuels, pharmaceuticals, and bioremediation) is examined in depth. Important extrinsic factors such as the impact of patenting and open source are also analyzed. With this information we feel we have laid the foundations for a comprehensive inquiry that will allow us to better understand what the expansion of synthetic biology in the business world will resemble.


    INDUSTRY ANALYSIS


    Before we attempt to understand the impact of synthetic biology on the business world as a whole, we must first understand what kind of an impact it will have on specific sectors. The goal of the industry analysis was to pick specific industries that we felt would be heavily impacted by the emergence of synthetic biology and attempted to understand their current situation as well as opportunities and threats facing each industry.

    The project wiki contains a condensed overview of the industry analyses, if you are interested in learning more be sure to check out the complete industry analysis.


    Biofuel Industry


    The oil industry has become an integral part of the society we live in for, transportation, food, healthcare, and communication. However, there is a dire need for alternative sources of energy and the world is beginning to turn towards biofuels for support in this area. Although there is immense promise in this area there is also many challenges to overcome such as reliance on environmental factors and adequate feedstocks. Although emergence of biofuels created through synthetic biology does not have the market potential to shift the whole method of biofuel production, politicians are optimistic that it has the potential to make an impact and have therefore taken measures to support the infrastructure of its’ development. The emergence of a viable synthetic biology biofuel would serve to excel the development of synthetic biology, particularly on the business stage.


    Pharmaceutical Industry


    Our knowledge of diseases and treatments has advanced to an exciting point as has society’s perception of health problems and issues. However, as fast as our knowledge advances, diseases like H1N1 and HIV provide a pressure for the pharmaceutical industry to advance further and faster. In addition, aging populations and growing urban centres poise pharmaceutical companies for significant and meaningful innovation over the coming decades. The pharmaceutical industry is expected to grow to an $800 billion industry by 2011, expanding as a global force. Thus, synthetic biology, although faced with challenges in terms of legal and social concerns, is poised to have a significant impact on the pharmaceutical industry. Not only is there promise for curative treatments that would change how we view illnesses like HIV, there promises to be significant implications in the social, technological, and political realms. Unanswered questions about the handling of intellectual property issues will challenge the development of synthetic biology in the pharmaceutical industry as will ethical, safety, and political issues.


    Bio-remediation Industry


    Unlike other industries, bio-remediation is an industry that has incorporated the use of genetic engineering and synthetic biology since the 1970’s. Based on the research conducted, there is a definitive understanding that this field poses an optimistic approach in achieving results that are environmentally friendly and cost-effective. The major setback with the field of bio-remediation is the intricacy of the techniques that are involved. Aside from the general publics qualms about bio-remediation procedures, even the scientific community does not have a holistic understanding of how the processes within bio-remediation work, or how to optimize a microbe’s “oil-eating” activity based on its metabolic characteristics. Most say that bio-remediation is a cost effective technique in treating vast oil spills, but it seems that investors and economists have not incorporated the cost of time and labour required in mastering the techniques involved. Once these techniques have established, there is a definite opportunity for this field to overpower the current invasive chemical processes that are being used as a last desperate resort in treating these environmental disasters.


    OPEN SOURCING and Synthetic Biology


    What is Open Sourcing?


    What differentiates synthetic biology from biotechnology is that it offers the creation of systems or pathways that would not be found naturally in an organism. Synthetic biologists believe in a standardized system of parts similar to that of electrical engineers having standard circuits and components1. This is also similar to how LINUX modules have been combined to create different software. This contrasts the closed-parts strategy where developers use such methods as patent protection and secrecy to gain a competitive advantage over others2. That is why the Massachusetts Institute of Technology (MIT) has created the initiative for what is known as the “Registry of Standard Biological Parts” where it indexes biological parts that are currently being built. A standard array of modular gene switches or parts that can be found from a common library and can be mixed and matched in various combinations, which is the goal that synthetic biology targets. This is a similar path as what happened twenty years ago when software became standardized and allowed Microsoft to become a monopoly. The issue in the air currently is if this could happen with the synthetic biology industry3.


    Advantages


    Open-sourcing has been an idea that has been the basic foundation of synthetic biology throughout the years. Supporters strongly believe in a world where companies and academia will be able to develop and share parts freely for the advancement of the whole field, not just a singular firm or university4.

    A term that is used commonly in this industry is called network effects. This means that the more a product is used, the more attractive it becomes. Over time as each part is used repeatedly on a specific metabolic pathway, especially when in successive experiments, its cost goes down. With the limited data that is available, it is predicted that total project costs could be cut down by 25% after its first successive use. It is also likely that these costs would be cut down several more times until it flattens as with each subsequent experiment more knowledge on that part is gained and intuitively, less errors are made.

    One such example is with Amyris’ artemisinin project that spanned over the length of five years, costing $20 million. It was reported that 95% of the time spent was on finding and fixing unintended interactions between parts.

    Due to the fact that one part must be used in conjunction with other sets of parts gives incentive to companies to create whole libraries. This is very similar to how software companies develop several programs that are able to cover multiple applications. Not only that, but there is opportunity for these companies to make profit by patenting some of these parts and making others openly available due to the strong modularity of the open-parts approach.

    It is expected that companies will have their own individual parts needs. This means that other companies cannot just sit around and wait for another to develop a part. This also gives a type of competitive advantage as companies that share parts will not be losing their, ‘technological edge’ to other competitors. Since different companies have idiosyncratic needs and hence expertise, it proposes that community-based libraries will outperform individual companies. The industry will probably have a large number of small, distinctive customers, meaning that patent licensing will be less attractive and the open-parts initiative more so5.

    Normally, the value of a patent depends on the inventor’s initial R&D investment, but with synthetic biology parts it would depend on that as well as how many researchers have subsequently used that part. According to the law, this allows patent owners to capture both sources of value, which can be unfair to society as they must deal with high prices without getting anything more in return. The open-parts initiative sets the price of parts equal to zero, so it is naturally able to solve that problem. There are two incentives that will draw companies to an open-parts initiative. The first would be the opportunity to share R&D investments among multiple firms and secondly the opportunity to produce parts faster due to shared insights6.


    Barriers and Disadvantages


    About twenty years ago when software became standardized, it opened the path for Microsoft to come in, take over the industry and monopolize it. Can something similar potentially happen in the synthetic biology industry? At the moment, synthetic biology is what is called a ‘tipping market’. It is unstable and prone to monopoly. Building on this, the tipping dynamic is indifferent to whether or not the dominant parts constellation is open or closed. If for instance a mature industry is able to grab a hold of these shared parts, it can work towards this open-parts initiative. Though on the other hand, companies, out of necessity will also pay for closed parts, both solutions are equally viable.

    Some other issues include an agreement on standard nomenclature for the parts, therefore when actually designing a database, controversies might come up. As well when collaboration occurs there must be adequate legal infrastructure. This must include a license specifying the rights and duties of members. One of the main legal problems is that gene data is unlike software, it cannot be copyrighted.

    Many pessimists towards the idea say that it is now too late for synthetic biology to use an open-parts collaboration. Now with Amyris’ advancement in the field it seems that this industry might monopolize and follow the path that Microsoft had. The fact that commercial synthetic biology receives so much government support shows a bit of laissez faire attitude. In America’s Department of Energy $350 million biofuel initiative there was no open-parts requirement at all. It is said that at least when Bill Gates cornered the software market he did it with private money7.



    1http://heinonline.org/HOL/LandingPage?collection=journals&handle=hein.journals/tlr85&div=50&id=&page 2,3,4,6,7http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726678/ 5http://www.nature.com/msb/journal/v3/n1/full/msb4100161.html

    PATENTS and Synthetic Biology


    The ideological reasoning behind applying for a patent lies in the fundamental incentive that it would bring forth financial rewards and a sense of monopolistic supremacy in an advancing market. The imposition of patenting technology has been deemed useful in many industries, most notably in electronics. However, it has also brought forth a considerable level of controversial dialogue in determining how historical protocols can be implemented within new technological streams that sometimes question the concept of man-made invention. For example, Craig Venter’s desire in protecting his synthetic cell research methodology gives him a deserving ownership, but his broad patent places a downgrading threat towards the field of synthetic biology. Ironically speaking, patenting is a legal practice that promotes innovation, but imposes a sense of apprehension for those who wish to adapt towards a more open-sourced strategy. This analysis focuses on the implications of introducing patent law to the field of synthetic biology as well as the practicality of gaining rights to a product not developed entirely by man, but with elements arising from nature.


    Monopolizing Synthetic Biology


    The accrued financial and proprietary benefit of monopolizing an idea within a competitive market is indeed an accelerated advantage when a patent is granted. However, many have the misconception that monopolistic proprietorship has a standardized value to any industry it is applied to. For years, Professor John Sulston from the University of Manchester, a believer in promoting an open-source environment in the field of synthetic biology has proposed the implications that would arise should Venter receive approval upon his protocol in developing synthetic organisms. Sulston states,

    "I hope very much these patents won't be accepted because they would bring genetic engineering under the control of the J Craig Venter Institute (JCVI). They would have a monopoly on a whole range of techniques (BBC UK, 2010)."

    The irony in this matter is that the technological industry used the concept of monopolistic competition to drive both innovation and the development of products that would eventually overpower the market leaders. The reason that this concept cannot presently be applied is that synthetic biology is strongly reliant on collaborative construction. Patenting biological parts or processes will not motivate a group of scientists to pursue their research, but cause them to weigh the opportunity costs between their groundbreaking research and the hefty payments they would make to patent holders.

    In the case of Venter’s claim, organizations such as the ETC group believe that Venter’s group is eyeing on a profit making opportunity. Hope Shands of the ETC group states, “The fledgling synthetic biology industry keeps talking about how they’re going to fix climate change – but these sweeping patent claims reveal that the companies are much more focused on securing profits than on human needs” (ETC Group, 2007). The multi-purpose use of a biological methodology would not only give Venter ownership to a scientific methodology, but to a range of applications that could be possible in the chemical, medicinal or environmental industries. Venter reported to Business Week, “If we made an organism that produced fuel that could be the first billion- or trillion-dollar organism. We would definitely patent that whole process” (ETC Group,2007). Many would dispute that this financial and market acquisition would only de-motivate other synthetic biology scientists and facilities to halt their research. But from looking at it from Venter’s perspective, wouldn’t anyone want to reap the rewards on a project that took 15 years and 40 million dollars to reach its success?


    Patenting Artificial Life: Is synthetic biology a man-made industry?


    As previously noted, synthetic biology encapsulates the field of biotechnology, software and computing. The current patent laws that have been put in place have generally been applicable to all industries individually. However, the field of synthetic biology is an amalgamation of two fields that have faced years of controversial debate in terms of granting or approving patents. The concern lied in the fact that patents related to inventions in biotechnology or software could be broad or narrow, but extensive enough to “hold-up” the concept of innovation and invention (Rai and Boyle, 2007 ).

    Synthetic biology is being renowned as a stream towards the development of artificial life, which for some raises social, ethical and legal concerns. Patent Act 101 states that any subject matter that is found in nature is not deemed as patentable. However, if the product found in nature is modified or transformed to something that is novel and non-obvious, it holds credibility in attaining a patent. Looking at the synthetic cell created by Venter’s team, it is difficult to denote the “man-made” material in his composition. His cell includes a computer generated “minimal genome sequence” that is encapsulated within a bacterial cell (found in nature) and still hosts cellular machinery required to allow the cell to adapt and function within its environment. The modification is within the genome, but even that raises some debate on whether it can be patentable.

    The software industry has always tackled with patenting mathematical and computational algorithms, particularly in concern was their level of broadness. In perspective to the field of synthetic biology, an algorithm is analogous to the creation of novel and modified genetic sequences, which give rise to new metabolic pathways and cellular functionalities. Yet again, the derivation of their new genetic sequence comes from the naturally degenerate genetic code, bringing up the question of whether this is entirely man-made. What is seen here is a disconnect, in that it would be difficult to set fundamental patenting protocols for the field of synthetic biology, given the legal complications faced in both the field of biology and computing. This is where many may agree that an open-source strategy such as the MIT Registry of Standard Biological Parts would bring forth more progression, versus the time and effort it would take to validate the patentability of a biological part or process.


    SWOT Analysis


    Below are other indicators to help define the relative benefit of introducing patent law, as well as the long-term threats that may hinder the advancement of research in the field of synthetic biology.


    Strengths


    •Patenting assists in stimulating investment, and secured investments bring forth progression in research. Given that research projects can cost up to hundreds of millions of dollars, patenting synthetic biology technologies would provide a more steady approach in financing the development of an invention.
    •Developers are motivated by reward, which is provided by the successful implementation of patent law Weaknesses
    •The field of synthetic biology is different in the sense that innovation is not promoted through attaining proprietary rights to an entire process or genome, but through the collaborative use of fine, functional and specific biological parts
    •Synthetic biology is multidisciplinary as it incorporates the field of biotechnology and computing, each has their own sets of legal rights, restrictions, and pitfalls
    •In addition, lawyers would require a more extensive level of knowledge and proficiency in both fields
    •Sometimes, hundreds of parts are necessary for the composition of one biological machine, and attaining rights to each part would create what is called to be a "patent thicket" (Rutz, 2009). Attaining rights to so many parts does not only hinder innovation, but is time-consuming and costly

    Opportunities


    • Patents add value, reputation and credibility to an invention. Although this legal concept is frowned upon in the R&D field, it brings a source of financial opportunity that would bring forth strategic partnerships and lump some funding to further progress research. At an economic perspective, patenting would be useful in advancing one's research.
    •Patenting requires thorough documentation and characterization of each aspect of the part, streamlined patenting protocols could add standardization to the part development process Threats
    •European patent law states that "iventions of commercial exploitation to morality are omitted from patentable rights" (Rutz, 2009). The general public fears that if rights to a biological process or part are give they may be abused. Examples are biological warfare or the unintentional release of pathogenic organisms.

    •"Patent sharks and trolls" (Rutz , 2009) may find it simple to file lawsuits against such patents. •On the other side of the coin, some have claimed that finding a patent application for a biological part is similar to finding a needle in a haystack. Moreover, it is not the job of a scientist to be rummaging around in identifying his legal rights to using a biological part, nor should his time be consumed by understanding the patenting process relevant to his field.


    Conclusion


    The concept of patenting makes scientists cringe at the thought that their research and advancements would be diminished by the monopolistic power held by the broad patent holders within synthetic biology. There is currently no established set of specific protocols for patenting products in synthetic biology. Moreover, the attempt to blend the patent laws applicable to a range of disciplines to one as intricate as synthetic biology is difficult. Additionally, companies who wish to have a formalized process in examining and submitting processes would have to understand that a large number of additional resources would be needed to accommodate for the labour intensive, time consuming and costly process of patenting such biological parts. So the underlying question is that what is of greater value in today’s market, leading innovation through open-source strategies or banking on profit-making opportunities by securing an idea with a patent?


    Conclusions and Outlook


    After conducting this research into the current and future position of synthetic biology particularly in the context of the business world, we are optimistic about the course that it will take. There are significant hurdles to overcome but overall there is a sense that synthetic biology will impart a positive impact not only in terms of specific products but also in terms of micro and macroeconomic impact.

    In this inquiry we have attempted to understand some of the important issues facing synthetic biology from a business perspective. In the future we hope to take our work a step further; we hope to take our knowledge, thoughts, and questions into the business world. Through interaction with business owners, scientists, legal specialists, and other stakeholders we hope to disseminate our findings and build on them. In the future we will continue to focus on our goal of deciphering the path that synthetic biology will take as it emerges in the business world.


    References


    1) Rai A, Boyle J (2007) Synthetic Biology: Caught between Property Rights, the Public Domain, and the Commons. PLoS Biol 5(3): e58. doi:10.1371/journal.pbio.0050058

    2) Barrett, Margaret. Intellectual Property. 2nd ed. New York: Aspen Online, 2008. 33-34

    3) Dickinson, Boonsri. "Will Patents Give Craig Venter a Monopoly over Synthetic Life? - SmartPlanet." SmartPlanet - We Make You Smarter - People, Business & Technology. 28 May 2010. Web. 26 Oct. 2010. .

    4) "Artificial Life: Patent Pending | The Economist." The Economist - World News, Politics, Economics, Business & Finance. 14 June 2007. Web. 26 Oct. 2010. .

    5) "Postnote: Synthetic Biology." Parliamentary Office of Science and Technology, Jan. 2008. Web. . 6) Rutz, Berthold. "Synthetic Biology and Patents : A European Perspective." Nature Publishing Group : Science Journals, Jobs, and Information. 2009. Web. 26 Oct. 2010. .

    7) Joachim, Henkel, and Maurer Stephen. "The Economics of Synthetic Biology." Molecular Systems Biology. Nature Publishing Group, 5 June 2007. Web. 26 Oct. 2010. .

    8) Ghosh, Pallab. "BBC News - Synthetic Life Patents 'damaging'" BBC - Homepage. 24 May 2010. Web. 26 Oct. 2010. .

    9) Ball, Philip. "Biology News: The Patent Threat to Designer Biology." BioEd Online. 22 June 2007. Web. 26 Oct. 2010. .

    10) ETC Group. "Extreme Monopoly: Venter's Team Makes Vast Patent Grab on Synthetic Genomes | ETC Group." 8 Dec. 2007. Web. 26 Oct. 2010. .

    11) Hammond, John, and Robert Gunderman. "The Limited Monopoly- Patent Law 101: What Is Patentable?" The Rochester Journal, June-July 2007. Web. 25 Oct. 2010. .
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    Parts List
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    UW's parts for 2010.


    BBa_K359002 - Signalling - Agr quorum sensing sensor/generator, FepA pore, with P2 + reporter
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    Safety:


    The health risks associated with the organisms used and the work completed could be classified as very low to moderate. Biosafety level 2 would be sufficient, as the organisms involved are Escherichia coli and Staphilococcus aureus. However, in practical use (e.g., hospital setting), level 3 facilities would be required. This is to account for several factors, including the increased potential for highly pathogenic strains in the vicinity, as well as the very unlikely possibility of a pathogenic strain of Escherichia coli obtaining the AIP sensing characteristics of our StaphiScope model.


    Facilities, equipment, and procedures which are required to contain risk group 2 organisms at Level 2 have been met:


    1.Laboratory separated from other activities.
    2.Biohazard sign.
    3.Room surfaces impervious and readily cleanable.
    4.Equipment includes an autoclave.
    5.There is personal protective equipment which includes laboratory coats worn only in the laboratory, gloves worn when handling infected animals.

    All precautions with respect to recombinant DNA are observed:
    All waste is autoclaved before being thrown away.
    Researchers practice aseptic technique, and frequent hand washing.
    Bench surfaces are disinfected with ethanol.