Team:Cambridge-JIC/Biolistics
£236.40
OUR LOW-COST, OPEN SOURCE GENE GUN: DEMOCRATISING THE TECHNOLOGY FOR CHLOROPLAST ENGINEERING
Gun Assembly
1. Following the method shown in the video below, apply PTFE tape to all BSPT threads - see table 1 for a list of the relevant hardware parts. I wrapped the threads 6-8 times, which seems more than sufficient.
Note: each time you unscrew a taped thread from the gun once it is assembled, you will need to remove the old tape and reapply following the method above before reattaching it.
| Part name | Side(s) to apply PTFE tape to |
|---|---|
| Schrader valve | ⅛” BSPT male |
| 1/4" to 1/8" reducer< | 1/4” BSPT male |
| Pressure gauge | ¼” male G thread |
| 1/4” male-male coupler (quantity = 2) | Both sides |
| Needle valve | Both sides |
| 1/2” to 1/4” reducer | Both sides |
2. Connect parts together for the upper half of the gun according to the diagram given in figure 1, and tighten the connections by hand.
3. Clamp the half gun in the vice and use the adjustable spanner to tighten each part until there is too much resistance to further tighten the connection. Note: do not overtighten the schrader-⅛” BSPT to ¼”-⅛” reducer connection (figure 2).
4. Unscrew the outer cartridge case on the CO2 pump (figure 3), and screw in a 16g threaded CO2 cartridge tightly.
5. UNDERWATER PRESSURE TEST (figure 4 - previous design shown) Fill your bucket with enough water to fully submerge the gun. Put the gun underwater, with the needle valve closed, and carefully open the pump as little as possible (the gas will come out very quickly when using a new cartridge). If the gun’s connections are sealed, you should only see bubbles come out the exposed end of the female tee. Try opening the needle valve after this, to check the outlet valve flow by observing bubbles coming out of the outlet.
Troubleshooting: if there are leaks in the gun, try tightening the connections again more thoroughly in the vice. Alternatively, check the connections are screwed in straight - if the ends are taped unevenly, the joints will be at a slight angle and will not be sealed properly. In this case, undo the connections, remove the old PTFE tape and re-tape the ends, taking care to wrap the tape evenly. Put the connections back together and then try pressure testing again.
6. Connect up the lower half of the gun, excluding the nozzle (refer to figure 1 again), and tighten using the adjustable spanner and vice as before.
7. Position the base onto the clamp stand and attach the clamps.
8. Clamp the gun to the stand - suggested positions for clamping are shown in figure 5. Adjust the height of the gun so its open end is approximately 5-8 cm from the stand base for initial testing.
9. Test firing without nozzle (I would recommend labelling the directions for the outlet valve as shown in figure 6, as a reminder when it needs to be closed quickly): If you are using a fresh cartridge of CO2, proceed carefully - the gun requires practice to fine tune reaching a desired pressure, and will be very sensitive at first. Wear eye protection and make sure the safety shield is in place around the gun (figure 7).
10. Open the outlet valve very slightly and make sure it is directed away from you and anyone surrounding the gun.
11. Very gently, open the CO2 pump handle as little as possible. If the pressure rises above 10 bar very quickly, turn off the CO2 supply then close the outlet valve. Briefly open and close the outlet valve until the pressure falls below 10 bar, to your desired firing pressure (recommended 100-140 psi).
12. If the pressure increases slowly (if you are using a half-empty cartridge, for example), you can close the outlet valve and keep the CO2 supply open until it reaches your desired pressure (still below 10 bar), then switch off the CO2 supply.
13. Ensure all hands/appendages are clear of the open end of the gun. Then, switch on the electrical box at mains and at the IEC filter (check the green SMPS LED illuminates at this point.
When you are ready to fire, depress the red trigger switch - there should be a fairly loud pop!
If the gun does not fire properly, you can release any remaining CO2 from the gun using the outlet valve - ensure this is also clear of bodily appendages before opening.
14. Nozzle composition (figure 8) - remove the rubber o-ring inside the nozzle, insert mesh filter into the hose nozzle, then reinsert the rubber o-ring on top. Cut a 5x3 cm piece of parafilm and place taught between two washers (figure 9). Trim the edges around the washers slightly, then fold any remaining parafilm over the sides of the washer and flatten (figure 10). Place the washer-parafilm sandwich on top of the mesh filter, then top with the o-ring in the nozzle (figure 11).
15. Rupture disc testing - repeat steps 10-12, then screw the nozzle onto the gun firmly in place (video clip 1). Make sure the safety guard is in place, then depress the red trigger switch on the electronics box. You should hear a loud pop of the parafilm rupturing.
16. Inspect the uniformity of the ruptured parafilm by unscrewing the nozzle (figure 12).
17. You can then experiment with thickness of parafilm (by stretching before sandwiching between the washers) and firing pressure, repeating steps 15 & 16 to optimise these parameters.
Bio-Makespace
Biomakespace is an initiative of synthetic biology scientists, students and enthusiasts in Cambridge who are working hard to build a new community laboratory. We aim to have a friendly sharing space where scientists could meet engineers, physicists, computer scientists, medics and other professionals but even public, students and schools. They all could start working together on synthetic biology projects from this academic year already. The iGEM team got involved with establishing of the space, planning and propagation from the very beginning.
A few of us are planning to share with other students what we have learned from synthetic biology over the summer by leading or participating projects based on cell-free systems there in the coming academic year under the flag of the new student-led Cambridge University Synthetic Biology Society
Emerging Biomakespace and similar community labs have also hugely motivated us for our hardware sub-projects as Biomakespace hasn’t initially considered working on plants and algae or their chloroplasts much. By offering our affordable hardware to them and similar community labs over the world we will facilitate further development of plant and algal synthetic biology and also work on chloroplasts. As an example biolistics is the only reliable way to transform chloroplasts of plants or algae. However costs of commercial gene guns are absolutely beyond what such labs can usually afford. We are offering a cheap and tested alternative opening a whole new range of possible projects for them.
Official aims of Biomakespace:
- Bring together biologists, engineers, technologists and others in the Cambridge area for meeting, co-working and socialising in a creative, cross-disciplinary, community-driven and safe environment.
- Provide a well-equipped space for practical biology and engineering of biology on a community membership basis.
- Support new and existing interdisciplinary collaborations for engineering biology, with a focus on promoting open technology and innovation.
- Raise awareness, understanding and participation in biology and engineering of biology in the Cambridge area through public engagement activities, education and training.
- Foster links with local industry and innovation organisations, building bridges between academia and bioenterprise.
OpenPlant
Understanding the bottlenecks of plant synthetic biology and best practices for open science
Hosted at the John Innes Centre in Norwich, the OpenPlant Forum presented talks from some of the most exciting innovations and research developing in plant synthetic biology at this moment. The three-day event also featured panel discussions on predominant issues in this field, including a discussion on “Commercial opportunities and bottlenecks in the future of plant synthetic biology”, featuring the inventor of BioBricks and ‘godfather’ of synthetic biology, Tom Knight.
This discussion raised the need for creating more efficient techniques for engineering plants into chassis for commercial production of biofuels and pharmaceuticals. This is something which we already aim to achieve as part of our project, through a standardised cas9 system for chloroplast engineering, which follows the phytobrick common syntax. Other relevant challenges were also raised in the discussion on “Reprogramming Agriculture”, such as responsible research and public perception of plant synthetic biology. Members of our team took part in this talk, sharing our views with an audience of over 100 people and making the key point that scientists have a responsibility to document their research and methods thoroughly. Mistrust and misunderstanding of what plant synthetic biology will be used for, and the ownership of this technology, is a result of miscommunication between the scientific and nonscientific communities.
The discussions highlighted the importance of DIY Bio Hackspaces, such as those we had encountered at the Bio NightScience event in Paris. By allowing ‘ordinary’ citizens to actively participate in synthetic biology projects for themselves, this helps to bridge the gap between the two communities and create a dialogue between them. This further encouraged our design of a low cost growth facility and gene gun for such spaces, as any techniques for plant engineering which industry hopes to commercialise must first be widely accepted in the public eye. Providing accessibility to these techniques for hackspaces, schools and other small community labs will, we hope, promote more widespread understanding and acceptance of them.
Furthermore, hearing Tobias Wenzel, founder of Docubricks, speak at the OpenPlant Forum gave us the idea to use this format as a template for the documentation of our open source designs. We hope that integrating the same best practices for open documentation used by this site into our own project will help to further support our efforts in removing the bottlenecks to chloroplast engineering, by changing public reaction and accessibility to the technology.
Synthetic Biology Society
Synthetic biology society is a group of students at the University of Cambridge aiming to bring together biologists, engineers, physicists, computer scientists and others to work in synthetic biology and share knowledge. It has been established by previous iGEM members in 2015. The society has ambitions to raise awareness about synthetic biology amongst students, broaden their knowledge through talks and as probably the only Cambridge science society it is actively working on primary research projects.
A few of our members got involved over the year with society’s activities and helped a bit with its beginnings and Lucie is on the current committee as the project manager. Last year’s project still in progress involves building a computer-navigated microscope moving in all three dimensions and scanning samples. Apart from continuing this we are planning a wetlab project in Biomakespace (have a link) which wants to explore cell-free systems. The project may change slightly but we want to make and tune a simple light oscillator (probably using fluorescent proteins fused to luciferase for sharper output changes) and also build a physical electric circuit imitating the biological system. From there we can study and demonstrate if the two systems behave and can be regulated similarly or differently, which may have a big educational value, we could also concentrate on transferring the system into a living organism or even some design using multiple oscillators. The emphasis in the project will be put on sharing skills, learning (even through seminars and trainings) and exploring what synthetic biology and scientific work in a team are all about!
coLAB OpenPlant
Investigating perceptions of plant biotech
In June, we spent three days taking part in the CoLAB OpenPlant Cambridge workshop, held in the University Plant Sciences Department. We engaged in and led discussions about the ethics of synthetic biology during the workshop, involving participants from 10+ countries with a variety of backgrounds, including art & design, medicine, music and biological sciences. As a part of the workshop, we learnt about the different techniques of investigating public perception and understanding, such as providing unexpected physical stimuli in typical environments and observing people’s reactions.
This culminated in us conducting our own surveys on the streets of Cambridge on deliberately thought-provoking issues surrounding plant biotech, to establish a dialogue with the public about plant synthetic biology and understand the opinions already held about this area. Some of the issues we investigated included the ethics of plant experimentation, comparing this with animal testing and seeing if similar emotional responses could be generated from questions such as ‘Do plants feel pain?’
We also investigated how people felt about potential future applications of plant syn bio, such as enhancing the nutritional content of certain foods, or even using engineered plants to express a person’s complete nutritional requirements which could then be extracted and concentrated into a pill-a-day form.
Although many of our questions were purely hypothetical, the conversations they generated were eye opening for us in understanding the reasoning behind some objections to plant syn bio. Participating in the workshop at this early stage in our project ensured we considered the impact of our work on different communities, and the potential reactions to the technology that could stem from the project, throughout the entire iGEM process.
