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Team:Dundee/Implementation/lasso
From 2014.igem.org
Hardware
The Real Thing
How it works
In the process of building the L.A.S.S.O. we optimized our circuitry in subtle ways to improve the photodetector’s sensitivity and reduce noise as much as possible. This was done in order for the readings of the samples to be as accurate as possible.
Since the first iterations of the biological devices use GFP and mCherry as outputs, we attempted to create a detector that would pick up fluorescence from these proteins. However, fluorescent proteins need to be excited in order to emit light which adds an extra degree of complexity. Therefore we considered a reporter whose nature eliminated the excitation step. Thus, we decided to work with luciferase, a bioluminescent enzyme, feeding back to the biological team that ultimately the biological devices would require luciferase as an output of signal detection. This meant that the step of excitation was removed and we would not have any issues related to filtering the excited fluorescence. Although light interference could be dealt with in our L.A.S.S.O., we also felt that this was the more reliable route to take in terms of our circuitry when building the remote monitoring sensor. Less components would also ensure that the L.A.S.S.O. could be kept less expensive and that the size could remain more compact.
Our final circuit uses an extremely sensitive photodiode. The diode has a peak sensitivity at 580 nm however it spans across the visible range from 330 nm to 720 nm which overlaps with the corresponding peak emission of luciferase which is at 482 nm1. It provides a range of readings for extremely low light levels. The circuit also contains a JFET Operational Amplifier2 as it is a high performance low-bias-current Operational Amplifier. This means that when it is used along with our high impedance photodiode the error of the input readings is minimized. The final major component of our circuitry is the section of low width bandpass filters. These are able to reduce the 50 Hz ambient noise created by mains electricity in the UK. We developed the idea of suppressing the 50 Hz of ambient noise that was interfering with our results further, by reducing it by a factor of 20 000. To minimize the noise in this way there are three low pass bandwidth filters in the circuit consisting of 100 kΩ resistors and 1 μF capacitors, which provide a time constant delay to the signal of 0.62 s (an improvement from our previous circuit constructs).
Building the L.A.S.S.O.
Since we had our optimized circuitry, we then moved on to building a case to house all of these components. We wanted to make sure that the L.A.S.S.O. was user-friendly and visually appealing. Essentially to make sure that future users, whether medical staff or patients themselves, could use the L.A.S.S.O. without requiring a set of complicated instructions.
When we first started building the L.A.S.S.O. we knew that we wanted our device to be completely blacked out from light. This was a technical aspect based on the sensitivity of the photodiode; it would prevent light from outside the box interfering with bioluminescent samples within the box. Hence, the only light that the photodiode circuitry would be picking up and amplifying would be the luminescence coming from the biological devices.
Other than the requirement to make the L.A.S.S.O. black, we had a lot of freedom in terms of the casing for the device. Knowing that it should be light and compact we decided to go with designing a structure ourselves. This way we could make all the compartments of our device as tiny as we wanted to, based on the dimensions of the circuitry components that we were already using. In order to execute the design, the L.A.S.S.O. was 3-D printed and the design came to life with three parts: one for the electronics, one for the sample, and the last one for a waste compartment. Since we wanted to make sure the device remained user-friendly we inserted ridges and corresponding depressions into the connecting parts on these three compartments. It meant that the whole L.A.S.S.O. could fit together easily and a new user would be able to see instantly how the three parts slotted together.
There are two major safety purposes why the device was designed to come apart - firstly for ease of access to the electronic equipment and secondly to implement a safety feature for the use of genetically-modified bacteria.
We envision the device being serviced every 12 months. This will ensure that the device is working to the highest standard. For this to be possible an engineer will need to have easy access to the detector’s circuit without damaging the L.A.S.S.O. To allow this we designed the lower section, where the electronics are housed, to be detachable from the top section. The electronics will then be housed under a platform which will have the photodetector positioned within so it is flush against the top. This will stop the lights on the Arduino from interfering with the detector readings and stop any wires from covering the diode.
The second reason is to create a safe enclosure for storing the sputum which has been exposed to The Lung Ranger. We want to minimise contact between the user and the biological devices as much as possible. To do this we have implemented a safety protocol for dealing with the samples. Once a user has finished reading the sample they are able to use a plunger attached to an arm to move it from the main device to a side compartment. This compartment houses a platform on a spring. As more samples are passed through they stack on top of each other and held in place, stopping them from moving around. Nine samples can be stored this way, meaning the L.A.S.S.O. can be used multiple times before it needs to be emptied. Once it does, the waste compartment can be removed from the rest of the device, and the samples emptied into special waste disposal. More information on safety aspects with regards to the L.A.S.S.O. can be found here.
We collected a preliminary set of results for the L.A.S.S.O. based on luciferase samples of increasing brightness. These samples were made through serial dilutions from a 5 ml overnight culture of Vibrio fischeri. A simple eye test was conducted in the dark room to arrange these samples by strength of luminescence (0 being the dullest and 5 being the brightest). A plate of media was also used to calibrate the results to record the background level of light present during the test.
| Plate Number | Voltage 1 | Voltage 2 | Voltage 3 | Voltage 4 | Voltage 5 | Voltage 6 | Average Voltage |
|---|---|---|---|---|---|---|---|
| Media | 0.321 | 0.319 | 0.300 | 0.303 | 0.309 | 0.311 | 0.306 |
| 0 | 0.340 | 0.342 | 0.343 | 0.341 | 0.317 | 0.319 | 0.334 |
| 1 | 0.423 | 0.418 | 0.363 | 0.366 | 0.356 | 0.362 | 0.381 |
| 2 | 0.390 | 0.387 | 0.345 | 0.357 | 0.342 | 0.337 | 0.360 |
| 3 | 0.457 | 0.455 | 0.444 | 0.443 | 0.398 | 0.404 | 0.434 |
| 4 | 0.507 | 0.503 | 0.462 | 0.460 | 0.438 | 0.430 | 0.460 |
| 5 | 0.589 | 0.587 | 0.562 | 0.567 | 0.526 | 0.522 | 0.559 |
References
1 Wikipedia (2014) Luciferase information [Online] Available from: http://en.wikipedia.org/wiki/Luciferase [Accessed: 30th June 2014]
2 Parker, G. (2004) Introductory Semiconductor Device Physics 216. New York, NY: Taylor and Francis Group
Welcome to Dundee's 2014 Wiki (Site Information)
Cystic Fibrosis is a genetic disease that results in the accumulation of thick mucus in the epithelial linings of the entire body, particularly the respiratory tract. Over the course of a patient’s life, the mucus-lined lung epithelium becomes repeatedly infected with pathogenic bacteria that stimulate an immune response; leading to inflammation, tissue damage, and ultimately, respiratory failure.
The microflora of the Cystic Fibrosis lung changes over time. In childhood, the major coloniser is Staphylococcus aureus, but as the patient matures other bacterial pathogens infect. The later-dominating pathogens, Pseudomonas aeruginosa and Burkholderia cepacia, are very difficult to eradicate and are associated with chronic decline in lung function. Burkholderia is so infectious that patients have to be isolated from one another, and can be denied lung transplants due to the persistence of this bacterium.
It is important, therefore, for medics to monitor and identify the levels of bacteria within the respiratory tract of a Cystic Fibrosis patient. Currently, patients must provide sputum samples and identification of bacteria takes between 72 hours and 2 weeks, by which point the bacteria can be in an antibiotic resistant state.
The Dundee 2014 iGEM project is focused on designing and testing a device that will rapidly and non-invasively identify the bacteria colonising a Cystic Fibrosis patient. Three biosensors will be developed that recognise external signal molecules produced by key bacteria, all of which are known to be in sputum samples of Cystic Fibrosis patients. A quinolone signal (PQS) is produced by Pseudomonas aeruginosa, a Diffusible Signal Factor (DSF) is produced by Stenotrophomonas maltophilia, an organism that is also associated with Cystic Fibrosis lung infection in adults, and BDSF is a related, but chemically distinct molecule that is produced by Burkholderia cepacia.
By engineering signal recognition and signal transduction proteins from these biological systems the Dundee iGEM team want to produce a portable electronic device that will identify infection outside of the clinic, so that patients do not have to travel great distances, and can be used to help health professionals make informed and rapid decisions on antibiotic treatments.
Read this again by clicking on Site Information in the navigation bar!
Bulletin Board (Policy & Practice)
Come look at the poster explaining how we involved the local and national Cystic Fibrosis community in our project, how we listened to people on the front line of the disease and how they affected our project.
School (Project)
Come in and learn about all the parts that make The Lung Ranger. The lab work that we carried out over the summer is explained here in detail, along with the modelling and implementation.
Library (Log Book)
Read about the experience the team has had over the summer. A week by week description of our work, how we carried out that work and some photos of our time together.
Saloon (iGEM community)
Come on in, take a seat and learn how we have interacted with the larger iGEM community. Who the team are, who helped them and how we helped others.
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