For our user friendly kit we required a circuit which displayed a simple on/off output once a certain level of DCM had been reached. The biochemists attached super-folder green fluorescent protein (SFGFP) to the promoter region of DCMA, such that when DCM is present, DCMA is produced and the bacteria have the ability to glow.

After initially assuming that GFP glowed on its own, the biochemists informed me that it needed to be excited by a specific wavelength of light before it could glow. For our project we chose to use SFGFP as it more stable than regular GFP. SFGFP has an absorption peak at 485nm and an emission peak at 520nm. Whilst there was plenty of data for GFP, SFGFP absorption and emission spectra data could not be found. As such we have used the spectra for EGFP which we believe to be very similar to that of SFGFP. As can be seen in figure 1, the absorption (blue) and emission (green) spectra cover quite a range of wavelengths with considerable overlap around the 500nm region. This overlap requires careful consideration in the design of the overall bio-detection kit as the excitation and emission light will mix and become indistinguishable to unfiltered sensors.

The configuration in our end product will follow that shown in figure 2. We will use blue LEDs to excite our SFGFP, they are ideal as they emit light in the range 450-500nm and are very cheap. Photodiodes are highly sensitive to the direction of the incoming light, so we will manipulate the circuit such that the LEDs lie at right angles to the photodiodes to reduce the amount of incident blue light. The mix of the blue excitation from the LED and the green emission light from the SFGFP will then be passed through a filter, which has the absorption spectra shown in figure 4. This filter will absorb lots of the blue light and little of the green light, so that a larger portion of the incident light on the photodiode is the signal we want to measure. We can calibrate our photodiode to ignore the small amounts of blue light transmitted through the filter, by taking light measurements whilst there is no SFGFP present.

To simplify the electrical analysis I will talk about the voltage levels at varying points throughout the circuit with two different light levels, light and dark.

A. When light is incident on the photodiode, a small voltage is created across its terminals. This voltage is immediately amplified by 10 to get a larger voltage at node A. (Using the BPW21 photodiode on the fifth floor of the Thom building, Vlight=4V and Vdark=-6V.) The voltage produced at A is now a sizeable amount but it is offset from 0V, which will make further analysis harder.

B. Using a voltage divider resistor network, we created a fixed voltage point equal to the dark voltage offset at A. We put an OP-AMP setup as a voltage follower between this and the next part of the circuit, to isolate the network and ensure that the voltage level created wasn’t affected by any circuitry downstream. This fixed voltage (-6V) can be measured at the node labelled OFFSET. We inputted the voltages A and OFFSET into a unity gain comparator. Which created a voltage at B which follows the equation B=OFFSET-A. By having the OFFSET equal to Vdark, it will cause the voltage for one of the light levels to be 0V. (Now at node B, Vlight=-10V and Vdark=0.)

C. If necessary we can amplify the voltage at B again using the non-inverting amplifier which has a gain which follow the equation: Gain=1+R10/R9. With the resistor values seen in figure 5, the gain=10. Although the maximum voltage we can produce is limited by the OP-AMP’s inability to provide voltages larger than the power rails which supply it (+/-15V). So by using the BPW21 no further amplification is available, so I removed R9 so that the gain was reduced down to Gain=1. So that at C we have Vlight=-10V and Vdark=0V.

D. For the final stage, we created another fixed voltage point which will control the threshold light level (-5V). Since the last OP-AMP has no feedback, the gain is effectively infinite and the output will saturate to the power rails. If the voltage at C is larger than -5V (Vdark=0), the output of the OP-AMP will saturate positively causing the voltage at node D= +15V. This will cause the green LED to illuminate, indicating that it is safe to pour down the sink as there is no light being produced, as there is no SFGFP present because there is no DCM left. When the voltage at C is lower than -5V (Vlight=-10), the output of the OP-AMP will saturate negatively causing the voltage at node D=-15V. This will cause the red LED to illuminate, indicating that it is not safe to pour down the sink as there is light being produce, as there is GFP present because there is still DCM left.

As desired we have a binary output which simply shows the user when the DCM mixture is safe to pour down the sink.

The two plasmids are partitioned during cell division by different systems, thus an equal proportion of each plasmid is maintained in each new daughter cell.

Different antibiotic resistances will allow us to select for cells that have taken up both plasmids by application of both antibiotics.

The replication origins compatible with E.coli and pseudomonas strains.

We have used two plasmids so that we can test each part in isolation before transforming them both into the same cell.