Team:Waterloo

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<h2>Our Team!</h2>
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<div class="teamProfile">Anum-ta Arif</div>
 +
<div class="teamProfile">Dan Barlow</div>
 +
<div class="teamProfile">Ekta Bibra</div>
 +
<div class="teamProfile">Diljot Chhina</div>
 +
<div class="teamProfile">Arpita Desai</div>
 +
<div class="teamProfile">Jon Eubank</div>
 +
<div class="teamProfile">Matt Gingerich</div>
 +
<div class="teamProfile">Behlool Khan</div>
 +
<div class="teamProfile">Leah Kocsis</div>
 +
<div class="teamProfile">Jordan Lapointe</div>
 +
<div class="teamProfile">Cveta Manassieva</div>
 +
<div class="teamProfile">Mariko Ozawa</div>
 +
<div class="teamProfile">Rob Pintwala</div>
 +
<div class="teamProfile">Brandon Wang</div>
 +
<div class="teamProfile">Fiona Whelan</div>
 +
<div class="teamProfile">Hilary Yeung</div>
 +
<div class="teamProfile">George Zarubin</div>
 +
 
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<div class="innerHeader"><h2>Our Advisors!</h2></div>
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<div class="teamProfile">John Heil</div>
 +
<div class="teamProfile">Eddie Ma</div>
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<div class="teamProfile">Andre Masella</div>
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<div class="teamProfile">Danielle Nash</div>
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Revision as of 00:44, 25 October 2010

UW iGEM Wiki

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.
Project
Modeling
Software
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:


1. Obtain sample from patient on cotton swab. This will, for example, be from a wound.
2. 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.
3. 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.
4. 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 deign 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 *INSERT TREE HERE*


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.


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.

Members
Staff
About UW

Our Team!

Anum-ta Arif
Dan Barlow
Ekta Bibra
Diljot Chhina
Arpita Desai
Jon Eubank
Matt Gingerich
Behlool Khan
Leah Kocsis
Jordan Lapointe
Cveta Manassieva
Mariko Ozawa
Rob Pintwala
Brandon Wang
Fiona Whelan
Hilary Yeung
George Zarubin

Our Advisors!

John Heil
Eddie Ma
Andre Masella
Danielle Nash
Parts List
Characterization
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2
Notes
Misc