Difference between revisions of "Team:NYU Abu Dhabi"

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 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:

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.

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:

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

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:

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?

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:

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:

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.

The results of the Interlab Study are tabulated here.