This section describes the hardware of our continuous microbiological culture device: TURBIDOSTAT.
What is the engineering aim of our turbidostat?
In order to perform a long-lasting in vitro demonstration of our proof of concept feedback system we constructed a turbidostat. This culture device ensures that the experiment conditions (cell density, temperature…) are maintained constant over time. Our Turbidostate allows us to keep a constant concentration on the producer and the reporter cells, as changes in the OD would affect the relation between lactone and superfolder GFP concentrations and, therefore, feedback could not be achieved in a consistent way. Of course such a useful device could also be used by other iGEMers for many other different purposes.
Where the inspiration for our turbidostat came from?
Living organisms exist in complex environments, which have certain conditions that favor their correct growth. Automated cell growth systems are capable of mimicking these conditions by adjusting variables such as temperature or Optical Density (OD) in order to maintain constant growth rates. Among them, there are DIY turbidostats such as the eVOLVER  that are composed of open-source wetware, hardware and software. In particular, our hardware design is inspired by the eVolver multi modular platform.
Maintaining the OD at a constant value is a key factor in these systems because it allows to have a rigorous and precise control of the living organisms inside, while not letting the cells get saturated and limited by the available resources.
What are the main foundations of our turbidostat?
Our hardware design (inspired by eVolver) mainly contains 3 modules:
When it comes to the first one of these three modules, the sleeve, it can be more deeply detailed attending to its four components:
- Aluminium tube: fundamental element to allow heat transmission from the external thick film heating resistors to the glass vial with the aim to accomplish the optimal temperature requirements of a cell culture to grow properly (about 37º C). Aluminum alloy 6060-T6 is chosen as the material for the tube due to its high thermal conductivity (~209 W/m*K) .
- 3D-printed piece (placed ringing the bottom part of the tube): its function is to house the 600 nm LED and its respective photodiode, both needed to know the OD of the culture inside the vial.
- 12V computer fan (placed at the bottom part of the sleeve): it is dedicated to keep the density of the cells homogeneous in the culture so that the values obtained for the OD are representative. To do so, a stirring magnet is introduced into the vial and two small disc magnets are glued at the top of the fan. Therefore when the fan starts spinning the culture inside the vial also spins thanks to the stirring magnet.
- Double methacrylate layer (placed between the fan and the tube): it separates the disc magnets from the bottom of the vial to avoid friction when the fan is switched on.
We invite you to see our user-friendly manual of a DIY turbidostat!
Finally, the electronics module together with the software part plays an essential role in the synchronization of the whole turbidostat. Information about this last unit can be found in the following section, Electronics module.
What about the electronic module?
In this section, a more detailed description of the circuits used for the management of the turbidostat can be found. Each subsection contains a schematic image and a brief description of the components. It has to be taken into account that, despite images here show a breadboard for the circuit integration, in the real implementation of the turbidostat stripboards are also used.
This first image shows the simplest circuit, which is just the fan directly connected to one of the Pulse Width Modulation (PWM) outputs of the Arduino. PWM allows the user to create analog outputs going from 0 to 5 V by introducing a digit from 0 to 255. In this case, since we do not need a really high power to move the fan at a speed that makes the stirring magnet rotate, no external voltage source is demanded. In fact, the analog digit used is 200, which implies using around 3.9 V. For our implementation the Qualtek FAD1-04020CBHW11 12 VDC computer fan is used.
When it comes to the circuit related with the OD measurements, we have two different parts:
- On the one hand, the LED is directly powered by one Arduino PWM output pin with around 3 V (limitation defined by the LED’s specification sheet). The LED that is used for our turbidostat is the Broadcom/Avago HLMP-C415.
- On the other hand, the photodiode is connected in series with a resistance and both are connected in parallel with a capacitor, which helps to manage voltage oscillations. The signal from the negative pin of the photodiode is taken to an Arduino input pin through another wire. The used photodiode is a Hamamatsu S13948-01SB.
Peristaltic pumps circuit
The circuit of the peristaltic pumps, like the one containing the heating resistores, is powered by an external source of 12 V due to the high demand of these loads. For each load (this is for each motor or peristaltic pump) a flyback diode is connected in parallel. This has to do with security reasons since the reverse voltage created when the motor shuts off can damage the transistor that controls the load if the current is not shunted. Moreover, a capacitor is also connected in parallel with the aim of filtering out some of the noise produced by the motor. These three components are connected to the drain of the transistor (middle pin). The right pin, which is the source, is directly connected to ground. Finally, the gate of the transistor (left pin) is connected to one of the PWM output pins of the Arduino, this regulates the current supply that goes to the load. The PWM output is connected to the ground through a resistance and to the gate through another resistance, these two resistances are optional. Of course the order of the pins can vary depending on the transistor, the one that has been used for our project is a MOSFET IRL540N. Furthermore, the peristaltic pumps that are used in our turbidostat have a 9 V DC motor
Thermistor-heating resistors circuit
This circuit, like the one of the LED & Photodiode, contains two different parts which are electronically independent but functionally correlated. Regarding the right breadboard, which is the one that has to do with the heating resistors, they are connected in series and their power is regulated by a transistor. The left pin of the transistor is the gate, being therefore connected to one of the PWM Arduino outputs. The right pin is the source, which is directly connected to the ground. Finally, the middle pin, which is the drain, is connected to the load (in this case the heating resistors) and the same protection components used in the pumps circuit. In the left breadboard, the thermistor is simply connected to the source line through a resistance and in parallel with a capacitor to absorb oscillations. Same MOSFET IRL540N transistor is used here. Regarding the heating resistors, they are TELPOD GBR-618-12-5-2 and the thermistor is an EPCOS B57863S0103F040.
Complete fluidic system
Is the development of a turbidostat the final step?
No, not at all. The final goal is to build a whole circuit of connections in order to prove the proper functioning of the feedback loop. However, due to several constraints (time, pandemics situation etc.) this has not been possible. Ideally, the hardware part should contain two turbidostats and another structure for sensing, thus one turbidostat would contain a culture of the producer cells and the other a culture of the reporter cells (please for further details about the cells visit proof of concept). These two turbidostats would be connected via peristaltic pumps with the third structure, which would receive lactone from the turbidostat of the producer cells and reporter cells from the other turbidostat. Since reporter cells produce sfGFP in the presence of lactone, the third structure would be devoted to sense this light and close the loop regulating the lactone to be produced. The amount of lactone can be induced by adding more or less arabinose to the producer cells or also by adding glucose that acts as a suppressor.
A sketch of the proposed implementation can be found below.
 Wong BG, Mancuso CP, Kiriakov S, Bashor CJ, Khalil AS. Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER. Nat Biotechnol. 2018 Aug;36(7):614-623. doi: 10.1038/nbt.4151. Epub 2018 Jun 11. PMID: 29889214; PMCID: PMC6035058.
 Aluminum Alloy . (2020, May 30). Retrieved October 27, 2020, from https://www.makeitfrom.com/material-properties/6060-T6-Aluminum.