Hi, we would like to share our progress (and ask for help) on a small project with some friends of the reGOSH/GOSH community (Nano, Nico Mendez, Fran Q, Wladimir, Seba, Severine, Valentina, Alejandro, Paola, Dominique, Tomas) and the Open Bioeconomy Lab—> using OSH for producing “cellular reagents” for low cost PCR in teaching labs or any other low resourced setting.
PCR reagents are expensive to obtain here and take a long time to arrive, etc (numbers here from BID/IADB report). Producing and purifying enzymes, as many of us do in Reclone, can reduce costs by 90% (we do it routinely in our labs). However, the expertise, supplies and infrastructure required to do protein purification is only found in well resourced research labs.
Very recently, a game-changing protocol from the Open Bioeconomy lab (extending the protocol from Andy Ellington´s lab) demonstrates that PCR reactions can be prepared directly from bacteria containing the PCR enzyme and grown+harvested from solid autoinduction media on petri dishes (link). Then, the PCR is performed using these intact bacteria containing the enzyme. So, bacteria are grown on solid media, collected with razorblade/glass slide, diluted and added to the PCR reaction. You use the bacteria carrying the enzyme as if you were using a pure enzyme. The use of autoinduction and solid media for the production of these “Cellular Reagents” avoids the use of expensive inducers (e.g. IPTG) and equipment for shaking/incubating liquid cultures. We took it from there with Jenny/OBL and all the people mentioned above to enable the implementation of this protocol with a low cost lab ecosystem:
- The use of the tupperfuge, a low cost centrifuge for cleaning the collected cells (it is required to remove pieces of agar and other “debris” from the bacteria harvesting step).
- OD measurement with an open colorimeter (ODI) after harvesting and washing the cells. This is an important step for deciding how much of the bacterial suspension needs to be diluted before using it in a reaction.
- The use of pocketPCR from GaudiLabs for the amplification reaction. We are big fans of this device. It works robustly everywhere.
- The separation of the amplified DNA by electrophoresis in a low cost chamber and open source power supply. HELP NEEDED: the power supply works very well but the gel chamber has been a pain. After trying different acrylic cases, the binding of acrylic was very laborious, trying to waterproof the 3D printed version was very difficulty and we ended up buying a cheap one on Ali and replacing the platinum wire. Thoughts very welcome.
- The visualization of results with an open transilluminator, which can be built locally in Chile/Argentina without depending on very specific and imported acrylics and LEDs. A nice collaboration with Jo from IORodeo here. We have worked with Fran Quero in a series of things:1) changed the superbrightleds that are very nice but expensive and needed to be soldered by hand, one by one. After trying different PCBs, Fran redesigned a new PCB with SMD blue LEDs that work very well and can be ordered already mounted. 2) replaced the acrylic filters with leefilters (used in the GMOdetective project). 3) replaced imported standoff and other bit by 3D printable pieces. With all these changes, the device works really nice and at a fraction of the original fabrication cost. We also add a usb-c alternative power supply because jack barrels are sometimes mixed up in a lab (e.g. 12,V, 15V, etc) and it is easy to burn things.
The protocol to prepare reagents and PCR using this kit is here (link) but in brief: grow E. coli containing the pTI vector (an open access vector we made in Reclone for protein expression (link)) containing a polymerase named OpenVent, fused to a red fluorescent protein (so you can see red fluor or pink color with naked eye, as a proxy for polymerase expression). The expression of these genes is normally controlled by IPTG (an expensive analogue of lactose metabolites), but autoinduction media do the job here since cells first consume glucose and then lactose, these metabolites activate gene expression. You incubate the plates at 37 overnight. We are testing bed blankets for this but any device that reaches 37C would do the job (e.g. kombucha fermenter mats, or close to an oven in winter). When the plates become pink, it means the enzyme and red fluorescent protein has expressed and you are ready to collect cells. Next, you have to collect the cells with a razor blade or microscope slide and collect them in an eppendorf. Here, you use Nico’s tupperfuge to precipitate the cells and wash any agar and debris carried during the harvesting step. Next, an open colorimeter is used to calculate the range of working bacterial densities. We are working on a method to measure red fluorescence with the transilluminator (same used below for the gel) and compare notes with OD-based measurements (sometimes bacteria growth does not equal good protein production, the two processes could be decoupled). After running the gel on electrophoretic chambers, it is visualized in a blue transilluminator.
We have successfully tested this ecosystem in many different workshops with more than 50 teachers and we use it routinely in our university courses (>100 students in total), it works but there are some Challenges ahead and Help needed:
- 37 incubator, we are testing electrical bed blankets and kombucha fermenter mats, etc but perhaps there is something better out there
- How to scale up hardware production/sale for users that don’t have the time or intention to build their own. A common issue. We know this is a very complex issues and already discusses in the community by many, but we leave it here because it is something we see a lot. At the moment, we are building all the devices (and buying Pockets and ODIs) and sharing them with a focus group of high schools teachers in Patagonia and teaching labs in universities as a first step/case study. But this is not sustainable if the demand increases.
Repositories (Ecosistema de Hardware Abierto para Biología Molecular – REGOSH):
- Taperfuga
- Colorimeter
repo & manual: Technical Overview - OLA is Open Lab Automata
Design inspired by:Open Colorimeter – IO Rodeo
- Pocket PCR
- Electrophoresis chamber
- Transilluminator
