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Revision as of 19:05, 16 September 2017

NYU Abu Dhabi





Project Description

The most common type of bacterial infection stems from contact with Escherichia coli, which when ingested can cause a variety of symptoms ranging from nausea to diarrhea. Shiga toxin-producing E. coli (STECs) are responsible for the majority of foodborne E. coli infections because the shiga toxin produced inhibits protein synthesis in all cells. While most countries now have stringent food safety regulations in place to prevent the sale of contaminated foods, small scale manufacturers, particularly street food vendors, often do not have access, time or pressure to consult laboratories about the safety of their food. Therefore, STEC-illnesses are still a major problem in countries that revolve around street food.

Our project aims to produce a portable device that allows for the detection of STEC through the use of loop-mediated isothermal amplification (LAMP), a technique that is similar to, but more sensitive than, polymerase chain reaction (PCR). The end goal of our project is to provide food vendors an opportunity to easily and quickly detect for the presence of STEC in their products to ensure that they are complying with government standards efficiently and conveniently. The results of each test will be uploaded into a database that provides consumers with the date, location and result of each STEC test. This will ensure that both vendor and consumer are safe, leading to a decreased incidence of foodborne E.coli infections.












Meet the team.












Major: Chemistry (Biochemistry)
Year: 2018
Where are you from: US/Taiwan
Something interesting about yourself: My spirit animal is a sloth.
Why do you want to participate in iGEM? I’ve always been interested in engineering and was curious to see how I could combine both passions.


Major: Biology
Year: 2020
Where are you from: South Korea
Something interesting about yourself: I’m a South Korean soldier next year.
Why do you want to participate in iGEM? You don’t say no to these things.


Major: Biology
Year: 2020
Where are you from: Argentina
Something interesting about yourself: I am interested in exploring what can be learnt at the interface between biology and other disciplines.
Why do you want to participate in iGEM? I think that iGEM is a great opportunity to develop an interdisciplinary project that draws upon biological knowledge to solve a real-world problem.


Major: Electrical Engineering
Year: 2019
Where are you from: Indonesia
Something interesting about yourself: I was 5 days away from getting struck by Tsunami.
Why do you want to participate in iGEM? Who says engineers can’t join a Biology competition?


Major: Biology
Year: 2018
Where are you from: Indonesia
Something interesting about yourself: In my life I’ve survived multiple earthquakes and volcanic eruptions… #Indonesian
Why do you want to participate in iGEM? My synthetic biology class introduced me to iGEM - I couldn’t wait to get hands-on experience in my final year!


Major: Biology
Year: 2020
Where are you from: Lithuania
Something interesting about yourself: Went to a 30km cross-country skiing marathon without knowing how to ski.
Why do you want to participate in iGEM? It seemed like a great opportunity to learn more about synthetic biology.


Major: Civil Engineering (Biochemistry)
Year: 2020
Where are you from: Pakistan
Something interesting about yourself: I have lived at ten different houses, each for at least more than a year, throughout my life.
Why do you want to participate in iGEM? To acknowledge myself with the meeting grounds of biology and engineering, and to learn how to integrate this knowledge in designing bioengineering systems.


Major: Mechanical Engineering
Year: 2019
Where are you from: Suva,Fiji
Something interesting about yourself: Not many people can find Fiji on the map.
Why do you want to participate in iGEM? YI get to work with machines and organisms, experimenting and creating a hybrid of life and automata.


Major: Mechanical Engineering
Year: 2019
Where are you from: Faisalabad, Pakistan
Something interesting about yourself: Snorkeled with Sharks, sea turtles and dolphins at three different reefs in the Maldives.
Why do you want to participate in iGEM? I’ve always had a love for biology and, well, biology and engineering together just sound irresistible so I couldn’t hold myself back.


Major: Computer Engineering
Year: 2020
Where are you from: Kathmandu, Nepal
Something interesting about yourself: I haven’t seen snowfall yet (I’m from Nepal).
Why do you want to participate in iGEM? The array of experiences required for iGEM, not only confined synthetic biology, instigated me to work on this project.



Development of a simple DNA extraction method

In order to use Loop-mediated isothermal amplification (LAMP) as the basis of the detection method of our device, it is necessary to include a DNA extraction step to obtain the template DNA from a sample.

We explored the use of heat lysis with different buffers to obtain template DNA from DH5-alpha competent Escherichia coli previously transformed with pGLO plasmid (BioRad). This extraction method is simple and could potentially be incorporated as an automated step in our device.

We prepared three buffers with varying compositions, adjusting their pH to 8.0:

  • Tris-HCl,
  • Tris-HCl + EDTA (TE)
  • Tris-HCl + EDTA + Triton-X 100 1% + Tween 20 0.5 percent

We diluted the bacteria culture to an OD600 of 0.6. 20 µl of this solution was mixed with 40 µl of buffer and subjected to 95 °C heating for varying amounts of time ranging from zero to 20 min. Using a NanoDrop™ spectrophotometer (Thermo Scientific), the A260/A280 ratio was measured (Fig 1). According to the manufacturer, a ratio of 1.8 is generally accepted as pure DNA. However, the presence of other cell components present in the sample may alter the results. The trials were ran in duplicates.

Figure 1. Comparison of A260/280 absorbance ratio of three different buffers shows no apparent difference between heating time.

Due to the poor results of the buffer containing Triton-X 100 and Tween 20, we discarded this option. Since it is in the important for the user to waste as little time as possible during the food test, we decided to limit the DNA extraction step to 10 min maximum.

The presence of template DNA was corroborated through PCR targeting a sequence in the pGLO plasmid. The PCR products were ran in an agarose gel. We used DH5-Alpha E. coli transformed with BBa_J04450 (backbone: pSB1C3) as negative control and pGLO isolated using the MiniPrep kit (Qiagen) as positive control.

The performances of the Tris-HCl and TE buffers were compared by running PCRs with template obtained through heat lysis at 95 °C for 10 min (Fig 2).

Figure 2. Tris-HCL and TE buffer PCR products with controls. 1) 1 kb+ ladder. 2) Negative control. 3) Tris-HCI treatment. 4) TE treatment. 5) Positive control.

Since the Tris-HCl treatment did not yield template DNA, it was discarded as an option.

Next, we used the agarose gel as a semi-quantitative measure of the PCR output to observe the differences in extraction performance due to varying heating times (Figure 3). We observed that the 10 min treatment had a clearer band compared to the other times. We decided to keep 10 min as our final time for heat lysis.

Figure 3. TE buffer PCR products with different heating treatment times. 1) 1 kb+ ladder. 2) Positive control. 3) No-heating treatment. 4) 1 min heating treatment. 5) 5 min heating treatment. 6) 10 min heating treatment. 7) Negative control.

It is still necessary to test this protocol with the LAMP reaction, which requires reagents that were unavailable at the time of these trials.

The Engineering Team

Meeting 2:


The initial design that we looked into consisted of one tube with two or three compartments. Each compartment was to be adjusted with a heater, so that the first compartment is at 95 degrees, and the second at 65 degrees. The third compartment already contains the color assay reagents that would then be used to give a color output to show whether if the test was positive or negative for the presence of shiga toxin. However, the small size of the sample brought into consideration a few issues, such as:

  • Evaporation of the sample
  • Excess dilution of the sample
  • Loss of sample as not all of it would pass on to the subsequent compartments
  • The fact that fluids rise as they are heated and this would prevent the horizontal or vertical flow of the fluid sample, as convection would always oppose the direction of preferred flow.

To resolve these issues, it was thought of to include a mechanical pressure system that would force the fluid sample into the direction of the subsequent compartments. A system of syringe was one of the methodologies that were thought of. Another method thought of was to use a capillary tube that would transport the fluid sample by a capillary force into the desired compartments. However, in using a capillary tube, the compartments themselves would be in the form of small separate tubes, all connected by capillary tubes that work on the basic principles of pressure.

Meeting 3:


The previously thought of ideas were discussed more in the light of theoretical experimentation. Using a valve between the compartments was also researched upon, and suitable valve systems compatible with various tube systems were thought of. Some novel ideas for the device also came up. These included:

  1. Using straws or any similar polymer material for the transfer of sample from one compartment to another, so that the valves can easily be integrated into the design.
  2. Using one heater device that can be automatically adjusted for the specific temperatures and the amount of time the heating device needs to be at that temperature. The tube would thus just have to be pushed a little so that at one time, the compartment that needs to be at the specific temperature is being heated.
  3. Using a branching system of flow of the sample. This works in the way that a large sample is pushed out of the first compartment, and as the tube that carries this sample is branched into smaller tubes, these smaller tubes make the liquid flow with a greater speed. Some of these smaller tubes take the sample into a “waste” compartment, and the others take the sample to the second compartment, where the subsequent reactions occur.

Request for the materials needed for experimentation was sent out so that we may begin testing our theories.

Meeting 4:


The 3D printed designs for the device, that were made as a result of the previous ideas and sketches, were assessed in terms of the microfluidic flow and the heating of the chambers.

The first 3D printed design works on the principle of applying physical pressure to push the fluid sample. It has four different compartments. The first compartment has the largest area, and this is where the sample is collected and heated at 95 degrees Celsius. The sample is then pushed forward. Among the next three compartments, two are waste compartments. This is where the excess sample is collected. And eventually, the last of the sample collects in the fourth chamber which is divided into partitions. This chamber is at 60 degrees Celsius, and this is where the lysis reaction takes place.

The second 3D printed design works on the principle of gravity. It is structured in a way that the sample would keep sliding down and then some of it will be collected as waste. The rest of it would overflow into the partitions where the lysis reaction will take place. The temperature for the initial sample chamber will be 95 degrees Celsius, and the temperature for the reaction where the lysis reaction takes place will be 60 degrees Celsius.

Moving on with these designs, the aspects of the device that need more research are:

  1. Inserting Valves: Both the designs need to be fitted with valves to control the flow of the fluid from one chamber to another. The material of the valves, and their design that fits accurately in the device cartridges is what the experimentation and research needs to be focused on.
  2. Inserting Heating SystemsSince two different chambers need to be at two different temperatures, appropriate heating systems need to be thought of. Currently, the ideas for heating are in the form of:
  • Using a water-bath or an oil-bath.
  • Using chemical hand warmers to generate the heat.
  • Asking a heater company to design heaters that can adjust to the device.







Introduction


The Fourth International InterLaboratory Measurement Study seeks to establish a reproducible plate reader-based GFP measurement protocol. Eight plasmids were transformed into competent DH5a E.coli cells and expressed for varying lengths of time. The optical density and fluorescence of each device was recorded over a 6 hour period. The major goal of this collective effort is to answer the following question: how close can the numbers be when fluorescence is measured all around the world?

Materials and Methods

See our extraction method here.

In this Interlab study, the following 8 RBS devices were provided in the Kit Plate 7 in the 2017 Distribution Kit:

  • Positive control
  • Negative control
  • Test Device 1: J23101.BCD2.E0040.B0015
  • Test Device 2: J23106.BCD2.E0040.B0015
  • Test Device 3: J23117.BCD2.E0040.B0015
  • Test Device 4: J23101+I13504
  • Test Device 5: J23106+I13504
  • Test Device 6: J23117+I13504

The transformation protocol was adapted from the iGEM protocol, which can be found here . All of the devices were transformed and cultured in LB (Luria Bertani) media containing 25 mg/mL chloramphenicol.
The following adjustments were made to the protocol:

  • 2 uL DNA were added to 50 uL competent DH5a
  • The incubation times on ice before and after the heat shock were 20 minutes and 2 minutes, respectively.
  • The heat shock was performed for 90 seconds instead of 50 seconds.
  • 800 uL SOC broth were added to each transformation.
  • Test Device 3: J23117.BCD2.E0040.B0015
  • After the one-hour shaking incubation, each transformation tube was centrifuged for 1 minute, 13,000 rpm. Afterwards, 700 uL supernatant were discarded and the pellet was resuspended with the remaining 100 uL before being plated onto LB agar plate.

On the next day, two colonies were picked from each plate and inoculated into two 5 mL cultures, yielding 16 cultures in total. The cultures were subsequently incubated at 37 ºC and 220 rpm for 18 hours. Afterwards, the OD600 of the overnight cultures were measured using a spectrophotometer. The cultures were diluted to OD600 of 0.02 in 12 mL LB medium and subsequently incubated at 37 ºC and 220 rpm. At t = 0, 2, 4, and 6 hours, 500 uL sample from each culture was saved on ice, yielding 64 samples in the end. The dilution calculation can be found here.

The 64 culture samples were transferred onto a clear, flat-bottomed 96-well plate according to the layout found here.
The OD600 absorption and fluorescence spectroscopy were measured using Synergy H1 Hybrid Multi-Mode Microplate Reader. The machine was previously calibrated using LUDOX. A fluorescein standard curve was also obtained under the same settings that were used to measure the culture samples.

Results

The results of the Interlab Study are tabulated here.