PENN iGEM 2015
IS THE LIGHT PRODUCED BY THE SENDER CELL SUFFICIENT TO ACTIVATE THE RECEIVER CELL?
In order to effectively sustain communication, the receiver cell must be able to respond to light and produce a reporter. To build our receiver system, we transformed the pDawn plasmid into DH5-α competent cells.
Light-sensitive pDawn contains YFI and FixJ genes that are preceded by the constitutively expressed LacI promoter. YFI is known as a Histidine Kinase; in the dark, it phosphorylates FixJ. Hence without light, FixJ binds to the FixK2 DNA binding site which is then phosphorylated. This induces the RNA polymerase to transcribe the lambda repressor which represses LacZ producing no luminescence. With the addition of light, the YFI and FixJ interaction is repressed, allowing the expression of the LacZ gene.
CONVERSION FROM RELATIVE TO ABSOLUTE LIGHT UNITS FOR RECEIVER ACTIVATION
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 of this device being known, as the inside mechanics are extremely complex and consist 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.
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.
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)
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, and as a result all RLU readings were normalized by their specific volume in uW.
DERIVING INTENSITY PER CELL
With the above conversion at our disposal we now were able to calculate something even more useful than the total intensity of the cultures we grew, the intensity given off per individual cell of bacteria.
By placing only 2uL of luminescing bacteria into a PCR tube, we were capable of treating this source of light like a point-source, in other words allowing us to assume that all of the bioluminescence created by the bacteria is being read by the detector. As a result, by calculating the number of cells in a given volume of bacteria, with the previous assumptions, we are able to derive the luminescent intensity of an individual SY104 bacteria cell.
Average Singular SY104 Cell Intensity = 3.23 x 10-9uW/cm2
The next step was to characterize the behavior of the team’s receiver circuit, pDawn-mRFP grown in a DH5A E.coli strain. There were 2 main important results came from this experiment:
- We were able to see that the receiver is a very sensitive piece of circuitry, thus capable of activating at very small intensities
- We were able to determine if our sender cells’ intensities were sufficient enough to activate the receiver cell.
Both these results were found by inducing pDawn-mRFP with external blue light in an aluminum covered MaxQ 445 Shaking Incubator. The blue light was produced by a series of blue LED strips taped to all four sides inside the incubator. This sort of set up allows the system to closely represent the uniform and equivalent delivery of light that is experienced by the receiver when it communicates with the sender shown in the figure below.
The intensities chosen were based off of the values of the SY104 strain. We approximated this strain's intensity by taking the average value once steady expression of luminescence is reached. As a result, the intensity calculated of an individual SY104 cell was used to approximate what the intensity of that culture would be if it 10mL was used to induce the receiver. We calculated this value to be approximately 8 uW/cm^2 and performed the experiments with a range of intensities within which the approximate bacteria intensity falls. The following data was procured: