Team:DTU-Denmark/Hardware
Microfermentation platform
When taking your synbio project to real applications, fast growth is crucial for viability. We took the concept of a DIY spectrophotometer and expanded it to a device that carries out three small scale fermentations simultaneously. With the files provided below you can build your own microfermentation platform, to compare the performance of our modified strain against a wild type or evaluate different growth media.
Overview
"When it comes to software, I much prefer free software, because I have very seldom seen a program that has worked well enough for my needs, and having sources available can be a life-saver."
Linus Torvalds
An important part of our project was the screening for substrates All this data was generated using a Hamilton robot for liquid handling. This robot enabled us to find the right organism to degrade our oily waste product. Such experiments are generally a great tool, to validate the growth on particular substrates or the degradation of toxic compounds. Furthermore, they can be used to compare different strains. However, we realized that it is a technology not everyone has access to.
To challenge this, we started a hardware project aiming to develop a cheap alternative to the Hamilton robot. A small device, that would enable hackerspaces and highschoolers to easily acquicire and compare growth data for their projects. To make this device as cheap as possible, we reduced the Hamilton to its key features:
Requirements for our robot
- OD-measurements
- Aeriation
- Stirring
- Automatation
- Data-logging
In order to optimize this prototype for our prospective users, we imposed these additional requirements:
- Simply reproducable from online material
- Development using only free soft- and hardware
- Modular and expandable design
Results
We build a working prototype that:
Theory
Growth rates and spectrophotometry
Basic concept - measuring OD
When assessing growth of microorganisms, it is necessary to be able to obtain and interpret information about population size, or at least concentration at a given time. Optical Density (OD\(_{600}\)) measurement is a widely used, suitable method known by everyone familiar with biotechnology. This principle is based on spectrophotometry, where a light transmission of 600 nm passes through biological material and the resulting transmittance, \(\Phi\) is measured at the receiver end. Because biological material reflects light, higher biological density reflects more light, giving lower transmittance readings. Thus, at full transmittance, \(\Phi_{\text{ref}}\), OD\(_{600}\) should be equal to 0.
For the Microfermentation Platform, OD\(_{600}\) at time t is calculated from the Lambert-Beer law REFERENCE: $$ \text{OD}_{600,t} = -\log\Bigg(\frac{\Phi_t}{\Phi_{\text{ref}}}\Bigg) $$ Where the light source is an LED, with its intensity controlled by some input voltage, and the transmittance is measured by a sensor and recorded as a voltage.
Growth curves of microorganisms
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From Spectrophotometer to Fermenter
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User testing
This device is meant to go out to fellow iGEMers and Hackerspaces. So intuitively we brought it to our friends from the local hackerspace Biologigaragen and our highschool student Tobias
Higschool student: Tobias
Tobias had no problems following the protocol we wrote. He liked the menu and the overall design. However, he mentioned that it was difficult to take the cuvettes out again from the robot because the holder is embedded very deeply in the housing. He helped himself by removing a panel and accessing the chambers from the side. A future prototype might be build slightly larger in order to increase comfort.
However, we also detected one major flaw. The protocol given to him did not include a point about regulating the airflow and the pump was only switched on while running the fermentation. Therefore he did not adjust it, causing most of the liquids to spill out in the housing and causing the program to crash. Consequently we adapted the protocol and the program so that the airflow could be regulated correctly prior to fermentation.
Hackerspace: Biologigaragen
Proof of concept
During the development, the design had to be tested several times and the results are shown there. After testing the protocol with Tobias, we ran a final test to prove that the overall concept is working now. The results are shown below
Prototyping
The prototype was build using laser cutting, 3D-printing and printed custom boards (PCB). It is controlled by an Arduino UNO R3 that was programmed using the Arduino IDE. All the files are created in the native formats of open source programs. We have used the following programs and recomend them, as the source files are in the respective native formats. In addition, all these programs have great communities to help, if you get stuck.
Software
- Inkscape to create vectorgraphics for laser cutting
- FreeCAD to make CAD drawings for 3D-printing
- ArduinoIDE for programming the microcontroler
- KiCAD for PCB design
Source files
The source files can be downloaded as compressed folders(*.zip):
History
This is a brief overview of the development history. For a chronological order, see the notebook
Starting point
Together with Erik and Martin we developed a first prototype. In this version, a single cuvette was stirried with a small magnetic pellet in a black measuring chamber. The light intensity was regulated manualy using a very precise geared potentiometer. The microcontroler received the voltage level from the photodiode, converted it into a 10bit number and send this via USB to the laptop whiche wrote the output to a file.
We learned the following:
- The LED remains constant during the whole duration of the experiment, giving constant light output.
- The Arduino requires an external power supply > 5 V for stable voltages
- Magnetic stirring in a cuvette is insufficient
Martin changed the container to a small glas flask which was giving sufficient stirring and demonstrated that the measuring unit was functional. However, the measuring cell grew even bigger. In order to reach our initial goal, we continued working on our own. We wanted to keep cuvettes as containers because they are cheap and easily available. Also our design needed the following changes
- No magnetic stirring
- No manually controlled potentiometer
- Data logging on µSD-card
New concepts
We could demonstrate that aeriation via common disposable syringes and needles was possible and sufficient to homogenize a S. cerevisiae culture at a tolarable amount of foaming.
In addition, we managed to replace the potentiometer by a set of low-pass filters combined with the 8bit PMW output-pins of the Arduino. This cut down the cost for the potentiometer and enabled us to use an automatic calibration routine to find the level of saturation for the photodiode.
We designed our own measuring cell. Compared to Martin's version it has a simpler locking mechanism to hold LEDs and photodiodes in place. It is also much smaler, takes three cuvettes and has connectors for aeration.
After debugging our circuit on breadboards, we designed printed circuit boards. Ten boards only cost US$ 10, plus shipping.
Make your own
We built this prototype with the purpose to share it with hackerspaces, highschools and other iGEM teams world wide. We cannot afford shipping devices all over the world but with the files below, you can recreate your own device, you will only need the ArduinoIDE out of the softwares mentioned before.
In principle, you can send the files out to different manufacturers and they will ship you the requested parts. We recomend this option for the circuit board, because US$ 16 for 10 copies is very good value for money. Especially if you have never soldered before, you might need one or 2 boards before you get a feeling for it.
For laser cutting and 3D-printing, we recommend you to ask around first. Especially 3D-printers are increasingly popular and the next fablab might be closer than you think. This will not only save you some money but bring you in contact with other makers.
Downloads
The source files can be downloaded as compressed folders(*.zip):
Acknowledgements
We have worked very hard this summer to create this device and achieved something that seemed totally out of reach only a few months ago. This would not have been possible without the help of other people. We would especially like to thank our supervisor Chris and Martin and Erik to pushing this project into the right direction. To see who else helped us along the way, see the full credit on our Acknowledgements page
