Why is this important?
We had three cultures: E. coli producing bacteriocins, wild-type E. coli (our pathogens) and cyanobacteria Synechocystis sp. PCC6803. E. coli grows on Luria Bertani broth at 37ºC, Synechocystis grows on our fertilized media at 25ºC. Bacteriocins kills enterobacteriae, but we also wanted it to be isolated from Synechocystis. So, cultures could not be mixed, but bacteriocins had to go from E. coli producing bacteriocins to wild-type E. coli and had to be affected by pH variations, caused by Synechocystis. You get the problem, right?
How have we done this?
We have drained the smartness out of our engineers in order to have a Do It Yourself system where all parts of our projects fit and work. This had its conceptual and practical problems, as you can imagine. Following is an animation of how it works, but please feel free to dig into it down below.
What are the requirements of the system?Every year iGEM comes up with dozens of projects with interesting applications, but few of them end up in a prototype. This year Valencia team has gone one step further building a scale prototype for a real application: disinfecting pathogens from water. The final design of our model has come after many hours struggling with ourselves in order to find the cleverest solution.
The prototype and its corresponding final design fulfill several requisites:
• Safety – The most important requirement, it has to be safe for the users and the environment
The team has worked much to avoid operating hazards and risk during construction. First, the corresponding safety measures for doing hardware work, like wearing gloves and using protection glasses were followed. Regarding the operation, it was decided to work with a 12 V energy supply, so there is no electroshock risk. Electrical cables have been protected and have been enough distantly kept from the water flasks. All other hazards related with biosafety have been explained in the corresponding biosafety section (poner link)
• Cost – Economically accessible for the majority of the applications
The total cost for out model is just 150 € (see list below)!
• Size – The dimensions of the apparatus need to be coherent with the disinfected volume and it is desirable to be able to transport it easily.
The size of our prototype is 70 x 55 x 25 cm, it is easily portable in a suitcase and weighs 7 kg.
• Ease of use – No advance technical expertise to be required
Once the cultures are prepared, there is no specific knowledge needed, just wait for clean water!
• Spare parts – The repair or replacement of failed parts should be simple and safe
Most of the elements of our design can be easily found and quickly substituted.
• Autonomous – Renewable energy sources like photovoltaic power generation are ideal to obtain a sustainable supply of energy
With the exception of cell cultures replacement after some time of use, the rest of the equipment can work alone if energy is supplied. With a total estimated energy consumption of 40 W, several renewable energy sources are adequate.
1. Bacteriocin production system
In this section there are two streams. One flows in a permanent loop and contains the E. Coli resistant bacteriocin producer with own synthesized bacteriocins. The second one passes through the UF membrane, so it is rich in bacteriocins and other small particles.
- Peristaltic pump (PP)
- Module of Bacteriocins Separation (MoBS)
This kind of pump is perfect to work with biological cultures, since there is no direct contact between the cells and the mechanical parts of the pump and consequently, no cell membrane damage and no temperature increase is expected. The fluid is contained within a flexible silicone tube fitted inside a circular pump casing. This tube is compressed by two rollers attached to the rotor, pulling the fluid in one direction.
Our PP comes from a used hemodialysis machine with an out of order motor. We replaced it with a flamboyant Volkswagen Golf windscreen wiper motor. In order to assemble the wiper motor with the pump we used following additional material:
The motor is coupled with threaded rods to a reinforced steel plate, which at the same time supports the PP. Thanks to the threaded rods we can regulate the distance from the motor to the pump and the angle of the shaft. In order to use the original shaft of the pump, we welded it to a nut with the same metric threat to the one of the motor shaft, achieving a cheap functional peristaltic pump.
The aim of this device is to separate the bacteriocins from the E. Coli harvest. There are marketed ultrafiltration modules of around 1000 € but after several designs and trials, we found a fully functional low-cost solution.
Our final MoBS device consists of different brass piping fittings. The main element is a 1’’ pipe tee with two reduced elbows where the central exit of the tee has an amplifier fitting connected to a hexagonal screw top. On the inside top surface the 2 ’’ Ø UF membrane is supported and its outline part is glued to the metal with a thin layer of neutral silicone, sealing and fixing the membrane at the same time. Both elbows have another fitting to end in a threaded nipple with ¼ ’’ hose barb whereby the flow which doesn’t pass the membrane comes and returns to the E. Coli bacteriocin production flask through polyurethane tubes.
The UF membrane is a flat NADIR® polysulfone membrane which is chemical and thermal stable and efficiently working for a wide pH range. Its MWCO value is 20 kDa, so bacteoricin peptides are able to pass the membrane in contrast to other big molecules and cells that are blocked by it. The incoming and outcoming flow of the tee are perpendicular to the permeate so that depositions on the membrane surface are prevented.
The pump is switched off when the proper concentration of bacteriocins is reached in the contaminated water.
2.Controller of pH (CopH)
This is the section of the whole system that controls the activity of the bacteriocins. It connects the flask containing pH regulating cyanobacteria with the water vessel. CopH consists of a ½ ’’ M-F pipe union with and ending fitting for polyurethane tubes. Inside the pipe union a dialysis membrane has been included to permit small molecules and ion exchanges but avoiding the pass of big molecules, peptides and bacteria, allowing proton transient transfer along the tubes that link both flasks. In this way, the pH value of the system is directly controlled by the photosynthetic bacteria to permit or inhibit the peptides actuation.
The dialysis membrane is a SnakeSkin™ Pleated Dialysis Tubing with a 3,500 MWCO value. We cut the tubing dialysis to form a ½ ’’ round and flat membrane and put it inside the pipe union, held by two equal size toric joints. Contrary to the UF membrane, this type is active at both sides of its surface.
All tubes with the exception of the one that is compressed by the peristaltic pump are made of polyurethane, while the one inside the PP is softer, made of silicone, so that the rotor is able to squeeze properly this tube. All tubes have a ¼ ’’ Ø. In addition, we have included throttle valves to all tubes that link the different functional parts of the machine (that means after the UF membrane and before the dyalisis one) as safety elements to be able to regulate the flows. Finally, it is critically required to include a throttle valve in the tube returning to the bacteriocin flask after the MoBS to be able to achieve enough P for the UF membrane separation process.
The main equipment of the apparatus has been described above. However, there are several extra instruments required to allow cultures to grow and develop.
- Magnetic stirrer
- Temperature Control System
- Energy supply
The agitation of the medium is essential for a correct homogenization of the bacteria and the compounds dissolved in it.
Our device is built with a computer fan and a neodymium magnet coming from a hard disk bracket. We stuck the neodymium-iron-boron alloy magnet on the axis fan, so when it is switched on, the magnet is permanently in movement, creating a changing magnetic field. The fan is put inside a watertight box for electric cabling. A stir bar is used to create the agitation in the flasks without affecting the biological harvest. Linked to each fan there is a potentiometer with which the desired speed can be reached.
The photosynthetic bacteria need light to do photosynthesis and so to change pH. Our illumination system consists of the LED lamp of a flashlight coupled to a switch.
E. Coli needs a 37 ºC medium to grow up at the maximum ratio. The team built a thermostat which keeps this temperature constant.
In the following the different parts of the thermostat, including the control circuit are listed:
The operational amplifier has two inputs, the non-inverting V+ and the inverting input V-. Moreover, the output is different to zero when the inputs are non equal.
In this way we can check the resistance of the thermal sensor, a thermistor, at 37 ºC and set the variable resistance to this value. Hence when the temperature reaches our target temperature, we have the same inputs at both entries and therefore the output from the amplifier is zero, opening the relay and so switching off the ceramic resistance that is heating up the biological harvest. The whole ceramic device is covered by a copper casing and sealed with silicone so it is safe to put it directly in water.
After the initial heating up phase, the resistance will be switched on just when the temperature of the solution given by the thermistor is below the target temperature. In this case, the ceramic resistance is switched on by the relay again and it restarts heating up the medium.
Several trials were tested using solar energy provided by our photovoltaic module to show that renewable energy sources are a future option for many synthetic biology applications. In other experiments, we have utilized a PC power supply for the different instruments of the disinfection machine. Depending of the device we have use one of the three available voltages (12, 5 and 3,3 V):• 12 V: Peristaltic pump, Temperature Control System
• 5 V: Lamp, Magnetic stirrer
Much work has been done to avoid electrical related risks, like film covering of cable welded unions. All cables have been together put inside a water tightbox. Finally three plug terminals have been adapted to the energy supply and to the tightbox to facilitate the connections and making possible to transport the supply energy separately.
Total prototype cost
One of the goals of the project is to create a working prototype that can be designed and built with low resources. We achieved this goal as our model just costs 150 €! Moreover, all the components can be easily found or bought in different stores with the exception of the two different used membranes that can be purchased online.
Many of the elements below listed were received as donation, but though an estimated cost has been given as reference expenditure.
The elements that have zero cost were taken from scrap