- Wet Lab
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- Human Practices
In order to bring our Directed Evolution system to life, we needed the appropriate hardware to run our experiment. Since our genetic circuit links the aptamer's binding affinity to the expression of antibiotic resistance genes, we needed a continuous culture device that would provide constant selection pressure and would adapt the concentration of the antibiotics inside our bacterias' growth medium, so that their concentration would remain stable. Luckily for us, such a device exists! It is a variant of the chemostat, termed the Morbidostat, originally developed in 20131. However, when we asked for quotes on the parts used in the original publication, we found that they tallied to over 9000 euros, a price which was prohibitive for us and we imagine, many other iGEM teams. Given that variants of the Morbidostat that can facilitate experiments of Directed Evolution/Adapted Laboratory Evolution2-4, we took inspiration from several of them in order to create our own, much cheaper and completely open source version of the Morbidostat. To the best of our knowledge, this is the first time a Morbidostat is built in iGEM
There are several parts that need to function in tandem, in order for the Morbidostat to work correctly. First, we had to establish a feedback loop between the bacterial concentration in our tube and the concentration of the antibiotics in the medium. For that purpose, we used a simple Light Emitting Diode (LED) and Light Dependent Resistor (LDR) system. The LED emits light at a wavelength of around 600 nm and the LDR's resistance is elevated, the more light it picks up.This system immediately introduces the need for a way of shaking our culture, since we need the solution to be homogenous, if our measurements are to be robust to error. For that reason, we used a simple fan with two neodymium magnets glued on top of it, as well as a magnetic stir bar. Another serious problem we had to face, were we to measure our bacterias' concentration with photometry was ambient light. The resistor is not so specific as to only respond to light of a certain wavelength. In order to reduce ambient light, we designed and printed a custom 3D printed Tube Holder with holes for space for the LED, the LDR, the fan and of course the vial of the culture, so we could produce accurate measurements. Next, a way to manage the antibiotic concentration within the medium was needed. The antibiotic concentration itself is never measured, only the rate of bacterial growth it, therefore, a semi-qualitative way of inserting more antibiotics in the medium is used. Adopting iGEM Aachen's 20175 design,we 3D printed and put together 3 peristaltic pumps, one for medium without antibiotic, one for medium with antibiotic and a waste pump. The drug concentration is increased by adding a higher fraction of antibiotic containing medium to medium without antibiotic, up to a maximum concenctration which is equal to the antibiotic containing medium. The only criterion to increase this fraction is the bacterial population itself, which is recorded continuously. Of course, a controller is needed to coordinate all this processes and for that reason, our whole system runs on 1 Raspberry Pi.×
We understand that not all iGEM teams have the ability to spend thousands of euros in order to build a continuous culture device. In order to promote open source science, we sought to create a cheap enough version of the Morbidostat, while not making any sacrifices to accuracy. Most of the parts used can be easily found in an electronics and a hardware store and the equipment used is common household equipment, like a drill, screwdrivers, or a silicone gun. Below you will find a table with prices and the store we bought from. We believe the prices show negligible changes between regions.Electronics
Part Quantity Product Page Link Cost Raspberry Pi 3B+ 1 Link 41.9€ NEMA17 Stepper Motor 3 Link 37.8€ Raspberry Pi Ribbon 1 Link 2€ Raspberry Pi Cobbler 1 Link 3.9€ microSD card (preferably at least 16GB) 1 Link 3.9€ Medium sized breadboard 1 Link 4.2€ A4988 Stepper Motor Driver 3 Link 9.6€ 47 uF 50+V Electrolytic capacitors 3 Link 0.15€ 12V 2A Power supply 1 Link 10€ Bright Red LED (close to 600 nm) 1 Link 0.2€ 5mm LED holder 1 Link 0.15€ 300 Ohm resistor 1 Link 0.01€ Light Dependent Photoresistor 1 Link 0.2€ ADS1115 Analogue to Digital Converter 1 Link 18.4€ Raspberry Pi Fan 30x30x10mm 1 Link 2.6€ TIP120 Darlington Pair transistor 1 Link 0.5€ 8mm diameter x 1mm width Neodymium Magnets 2 Link 0.56€ 10K Ohm resistor 1 Link 0.01€ 4 cm Magnetic Stir Bar 1 Link 3.8€ Pyrex Vial 1 Link 8.2€ Autoclaveable Tubing 5 metres Link 10€ 3D printed parts Varies None 30€ Jumper Wires 140 Link 4.50€ Flanged Ball Bearings 4x8x3 mm 18 Link 25.2€ Straight pins 4x50 mm 9 Link 5.85€ M3 screws 16 mm 12 Link 0.72€ M3 screws 8 mm 12 Link 0.6€ 50 mm screws 9 Link 1.35
The cost of this implementation of the Morbidostat costs shy of 300€, a far cry from the one presented in the original paper, which cost about 40 times as much. There are of course, a few caveats. This cost is calculated for 1 culture device and it each culture device added adds about 60€ to the cost. Nevertheless, we believe that this open-source, modular and easy to modify version of the morbidostat can help many teams in their experiments, especially when dealing with Directed Evolution. Following iGEM Heidelberg's 20176, we want to make Directed Evolution techniques available to a wider community, as we believe the technique is very powerful.
While due to the iGEM schedule, we weren't able to fully characterise the Morbidostat by running a long term Directed Evolution experiment on it, we included Calibration Scripts, which we suggest running before the experiment. In the graphs below it can be easily seen that the pumps have variation between them, something that renders calibration very necessary. Those calibration scripts ensure that the software controlling the Morbidostat is running smoothly, pumping correct volumes and measuring the correct bacterial population concentration. While our system is quite robust, it is by no means perfect and therefore, we suggest running the calibration scripts every once in a while, to check for possible mishaps in the system constituting the morbidostat. There are two calibration scripts, one that calibrates the pumps and one that calibrates the LED LDR system. The script that calibrates pump activates them 10 times for 1 second and 2 seconds each and the user is required to input the difference in volume each time. If the standard deviation is above a stringent criterion, then the pump must be reassembled. As for the script that calibrates he LED LDR system, it works by creating a calibration curve. The user is tasked with bacterial populations of different, but known concentrations then the system uses an Ordinary Least Squares (OLS) method to fit the data, so that measurements on the LDR are translated to bacterial cells/mL. These scripts are of course included in the relevant repository.
Of course, this would not be an open source project if we did not include a building guide. The building guide is available as a .pdf file in the iGEM site by clicking here. We tried to make it as pictorial and as detailed as we could, however, due to our inexperience with making guides for such project, some elements might be confusing, unbeknownst to us. Interested readers are invited to contact us with suggestions on the guide, the build, the code or questions regarding any and all of the above. Along with the .pdf file hosted on the website, we will have the code needed and the schematics in our repository, which is the most likely to be maintained in the future.
- Toprak, E. et al. Building a morbidostat: An automated continuous-culture device for studying bacterial drug resistance under dynamically sustained drug inhibition. Nat. Protoc. (2013). doi:10.1038/nprot.2013.021
- Liu, P., Lee, Y., Wang, C. and Yang, Y. (2016). Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection. Journal of Visualized Experiments, (115).
- Wong, B., Mancuso, C., Kiriakov, S., Bashor, C. and Khalil, A. (2018). Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER. Nature Biotechnology, 36(7), pp.614-623.
- Gopalakrishnan, V., Krishnan, N., McClure, E., Pelesko, J., Crozier, D., Williamson, D., Webster, N., Ecker, D., Nichol, D., Mandal, S., Bonomo, R. and Scott, J. (2019). A Low-Cost, Open Source, Self-Contained Bacterial EVolutionary biorEactor (EVE).
- 2017.igem.org. (2019). Team:Aachen/Hardware - 2017.igem.org. [online] Available at: https://2017.igem.org/Team:Aachen/Hardware [Accessed 21 Oct. 2019].
- 2017.igem.org. (2019). Team:Heidelberg/Predcel - 2017.igem.org. [online] Available at: https://2017.igem.org/Team:Heidelberg/Predcel [Accessed 21 Oct. 2019].