Introduction

We are aware of the necessity of using hardware in synthetic biology. In our case, to obtain experimental data for our mathematical model, we required measurement of some parameters, these parameters are fluorescence and optical density. For this, we built an artefact that would allow us to measure these qualities, relying on the iGEM Aachen 2014 team. For environmental conditions, we consulted previous experiments on scientific documentation. We build this hardware considering both our current needs as equipment, as well as those of future equipment. Therefore, this hardware is affordable and easy to use. The total cost of the device does not amount to $100 US dollars. The hardware was programmed in Arduino and Python. Those codes are in the repository which were carefully commented on thinking about future iGEMers that could occupy them.

For the design of hardware, we relied on the device designed by the Aachen team from Germany in 2014. In addition, we researched parameters and conditions in which the optical density and fluorescence measurements were taken. To quantify the protein production of P. Pastoris, was proposed the development of an EGFP sensor, as we established a one-to-one relationship of protein production with the intensity of EGFP fluorescence. Of the papers consulted where there is previous experimentation, we have the following

Table 1. Wavelengths

That is why LEDs of 490 and 600 nm were chosen.

In addition, in the paper On-Line Green Fluorescent Protein Sensor with LED Excitation 2 types of measurements were made, on-line and off-line. On-line consisted of measurements taken through the sensor continuously every 2 minutes and off-line measurements made every hour with a fluorescence spectrophotometer. The conditions of the measurements indicated in the article were as follows: The GFP, having 2 absorption peaks at 395 nm and 475 nm, was chosen to excite the longest wavelength excitation peak. Aeration at 1 vvm (volume of liquid per minute), agitation at 300 rpm and temperature at 30 °C.

The hardware has two analogue light sensors OPT101 and two LEDs, each sensor is related to an LED to perform fluorescence (F) and optical density (OD) readings. A filter will be used as a screen between the medium and the sensor in order to block any light emitted below 600nm (orange filter).

In this way the sensor will be able to measure the OD600. For fluorescence measurement it is the same principle but in this case a filter (green) was used to block any exciting light other than the light emitted by GFP.

In addition, it will need an Erlenmeyer flask in which the sample of P. Pastoris will be inside, and there is a magnetic stir bar that will shake it without affecting the system. As indicated above, the device includes two sensors, which takes readings in real-time and send the information to be read by Python and shown in a plot, as data is collected continuously, the Arduino's memory can get saturated. For this, the information only saves the last measurement.

How it works?

Fluorescence

For fluorescence measurements the absorption of light with a specified wavelength is essential. Some substances absorb light at one wavelength, and then emit light called fluorescence at another wavelength. This is a phenomenon in which a substance absorbs light to reach a high-energy level and then emits light to return to its original level. A UV/UV-VIS detector detects light that has passed through the flow cell, an FL detector detects fluorescence emitted in the direction orthogonal to the exciting light. Fluorescence detection is suitable for trace analysis because of generally having high sensitivity and selectivity (not detecting impurities) (Hitachi, 2022)

Figure 1: Diagram for Fluorescence sensor

Optical density

The traditional method for culture density measuring involves measuring 600 nm absorbance of the culture relative to the medium (Myers JA. et al., 2013). This is named the optical density of the culture OD600. The light is not absorbed but is scattered and the relative turbidity of the culture is in fact measured. Though a turbidity sensor is a device which emits light, passes it through the culture and reads the light intensity received (Kiviharju K. et al., 2008) (Marinescu et al., 2018)

Figure 2: Diagram for Fluorescence sensor