Team:Penn/Receiver
PENN iGEM 2015
RECEIVER
As can be seen, the data describing the Sender circuit is in units of Relative Light Units (RLU’s) based off of the Tecan M200 machine used for luminescence output measurements. Without the geometry being known of this device, as the inside mechanics are extremely complex consists of a Photon Multiplier Tube attached to fiber optic, it was impossible to convert the given RLU values into the absolute unit with which the Receiver circuit was characterized, uW/cm^2. Ultimately, this creates a large dilemma in the data analysis, as both circuits are described in different languages.
Therefore, we worked to create a 2-step calibration of the Tecan M200 was performed which made it possible to freely convert between Tecan RLU measurements and uW/cm^2 equivalents.
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LUMINOMETER GLOMAX 20/20 RLU to POWER METER uW/cm^2 (RLUs)
The first necessary step, was to establish a relationship between RLUs and uW/cm^2 using light that can have its intensity measured very easily. With a Thorlab Power Meter it was very simple to place an LED (wavelength 480nm) near it and measure its given intensity. However, an LED was still not capable of being placed into the Tecan M200 as it would completely saturate the machine’s sensory systems and an LED’s wiring would not fit inside the device. As a result, the Glomax 20/20 Luminometer was used instead. This device has a much more simplistic design, allowing us to actually attach an LED of known intensity (previously measured with a Thorlab Power Meter in uW/cm^2) right above the luminometer’s detector.
However, it turned out that just like the Tecan M200 was completely saturated with an LED, the Glomax 20/20 presented us with the same behavior. To solve this complication, we sought to find a way to reduce the amount of light hitting the surface of the detector in the luminometer. It turned out that a dark-red light filter which cut out about 4500x the light hitting it was ideal for taking measurements of an LED in the closed luminometer.
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This allowed the luminometer to produce RLU values from the LED and all we had to do was multiply the resulting RLU value by 4500 in order to find the actual amount of lighting hitting the detector.
With this information it was possible to create the graph below showing the relationship between a luminometer measurement and its equivalent absolute intensity in uW/cm^2:
TECAN RLU to LUMINOMETER GLOMAX 20/20 RLU(RLUs)
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Now that we had obtained a method of translating the language of RLU into an absolute intensity value the next step was to figure out how to convert Tecan RLU measurements into Luminometer RLU and therefore allowing us to convert into uW/cm^2.
To complete this step, luminescing bacteria was used as the source of light as this was something that we were able to place in both devices. The bacteria was measured on each device with various dilutions (1:2-1:50) with a total of 11 measurements in total. The filter was still kept when the measuring the bacteria’s luminescence with the Luminometer, in order to be able to plug into the previous conversion. The sample volumes for each machine are different by nature of the technologies’ requirement, as a result all RLU readings were normalized by their specific volume in uW.
The light producing component of the communication system is initiated by a constitutive promoter (BBa_J23100) and followed by an RBS and the lux box. Expression of the genes in the lux box is responsible for producing luminescence. The box contains the following genes in order: LuxC, D, A, B, E and G. Lux A and B are responsible for encoding two subunits of bacterial luciferase. The genes LuxC, D, and E code for the substrate for the light emitting reaction, tetradecanal. The luxG gene has no known function, however, inclusion of the gene is known to increase light output. The circuit is completed with a stop codon and a terminator.
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