2-in-1 OD and Fluorescence Sensor (INNOVATION)

The 2-in-1 optical density (OD) and fluorescence sensor takes continuous OD and fluorescence readings of samples of the bacterial culture. These readings are feedback that will be used to control the activation of the LEDs in the fermentation chamber, and thus regulate metabolic flux.

Design

The integrated sensor consists of two structures - a cuvette through which bacterial culture flows, and a casing to contain the 2-in-1 sensing system and the cuvette. We capitalized on the fact that the emission wavelength of RFP (See Design: Stress Reporter), 600 nm, is the same as the wavelength at which the OD of a sample is conventionally measured.

Structure - Cuvette

The irregular shape of commercial cuvettes made an analytical understanding of the light paths passing through it difficult (Figure 1). Thus it was challenging to design a sensing system around it. Since a commercial cuvette is small, modifying it directly is also difficult.

We thus decided to design our own cuvette (Figure 2) using 1.5 mm thick acrylic sheets. Apart from allowing us to ensure that the walls are uniform, it also allowed us flexibility in the placement of the acrylic tubes channeling media through the cuvette.

Our cuvette was designed to have as few unique parts as possible. This was to facilitate easy assembly, which is an important design consideration since the cuvette is small. We would need to be able to quickly fabricate many cuvettes for testing.

This design choice proved useful when we were troubleshooting the cause of our cuvette’s leakiness. Acrylic glue could not form seamless bonds. Even if it appeared so from visual inspection, leaks sprung once we tested the cuvettes by pumping water through them at high pressure. After several rounds of trial and error with other adhesives, we discovered that Acrifix 1R 0912 UV adhesive formed a watertight seal and is clear when cured. We used it in our final design iteration for the cuvette.

Structure - Casing

The casing houses the sensing system, which comprises a TSL235R light-to-frequency converter, a 600 nm LED, a 535 nm LED, and a LEE filter with a peak transmission of 600 nm. (Figure 3). The 600 nm LED is in the slot opposite the TSL235R, and is used to measure optical density (OD). The 535 nm is in the other LED slot, and is used to measure fluorescence. The LEE filter needs to be cut to an appropriate size and attached over the TSL235R. We used masking tape to secure the filter.

Feedback Control

Will put flow chart

Testing

We validated the functionality of this component and characterized it by plotting graphs of sensor output frequencies against RFP readings measured using the NanoDrop. Our feedback control system does not require the simultaneous measurement of OD and fluorescence. Moreover, the different LEDs involved would not both be activated at the same time, and hence would not interfere with each other. We were thus able to calibrate OD and fluorescence separately.

Procedure

We used BL21*-Prep-RFP-YbaQ E. Coli for OD and RFP sensor characterisation and test. Actual readings from nanodrop for OD and microplate for RFP were taken along with our sensor readings.

First, we prepare 20 mL of BL21*-Prep-RFP-YbaQ E. Coli with OD 3 while blue light was always on to repress RFP expression. The cell medium was diluted to get 12 samples of different OD ranging from 0.1 to 3. The samples were measured with nanodrop to get actual OD reading. Each sample was then transferred into the cuvette with a syringe. OD of all samples was measured using our OD sensor. A 600nm red LED is used as light source. Readings from light-to-frequency converter is an indication of light intensity passed through the sample. Smaller reading means less light is passed through sample so it has a higher OD.

For RFP sensor testing, we prepared 20 mL of BL21*-Prep-RFP-YbaQ E. Coli with high red flourescence (7100 in our microplate reader). It was diluted to get 12 samples of different RFP ranging from 100 to 7100. The samples were both measured with microplate reader and our sensor. In our sensor, a 535nm green LED is used to excite red flurescence and same light-to-frequency converter is used to measure red flourescnece. Higher reading means higher flouresence.

We also demostrated the sensor with our whole bioreactor setup. It's shown in the bioreactor parts.

Results

Construction

It’s beautiful. It’s obscenely integrated. It’s something you want right now. So why don’t you make it? We’ll help you!

Bill of Materials

• Arduino Uno x 1
• 600 nm LED x 1
• 535 nm LED x 1
• Light-to-frequency converter TSL235R x 1
• LEE filter (peak transmission 600 nm)
• ID = 3 mm acrylic tubes x 1 m
• Acrifix 1R 0912 UV Adhesive

Structural Assembly