Team:Utah State/Notebook

1. Electroporation Transformation of E. coli:

a. Researcher safety?

1. Turn on ice machine 2. Thaw DNA solutions 3. Clean and sterilize the electroporation cuvettes by washing with double distilled water (ddH2O) twice and then fill the cuvettes with ethanol. 4. Let cuvettes sit with ethanol for 5-10 minutes, then wash 4-8 times with ddH2O 5. Place cuvettes on ice 6. Take competent cells out of the -80 °C freezer, and thaw them on ice 7. Add 3 µL of DNA to the cell solution. (This should be around 100-250 ng of total DNA, too much DNA causes arcing, too little gives few transformed colonies). 8. Incubate on ice for 5 minutes. 9. Add 60 µL WB buffer (10% glycerol). This helps reduce arcing, although too much can lower numbers of transformed colonies. 10. Set the electroporation machine to 2500 V, 200 Ω, and 25 µF for E. coli. 11. Transfer the cell/DNA/WB solution into the cuvettes by pipetting up and down in the 1.5 mL tube first to mix. Make sure the pipette tip is between the metal plates on the cuvette before ejecting the solution. Keep the cuvettes on ice. 12. Before electroporating, dry the cuvettes of with a KimWipe, to ensure no liquid on the surface that could create other paths for the electric pulse (and could cause arcing). 13. Pulse the cells and return cuvette to the ice. Check the time constant on the machine, a constant of 4.5+ is a very good transformation, and will yield many colonies. A constant of 2.5-4.5 is okay, and will still work. Constants below 2.5 will yield very low colony numbers, and may need to be redone. NOTE: addition of extra WB or lower amounts of DNA will reduce the time constant as well, so it is only a rough measure. 14. To remove the cells from the cuvette add 1 mL LB media or SOC media (no antibiotic in this media) to the cuvette. Pipette up and down a few times to mix. 15. Remove the solution to just above the two plates in the first removal pipetting (~1/2 of the volume) and transfer it to the original cell tube (NOT THE DNA TUBE). Then, tip the cuvette on its side so that the space between the plates is vertical, place the 1000 µL pipette tip between the plates, and slowly draw up the solution, while tipping the cuvette further. This should draw up the rest of the liquid in the cuvette. 16. Incubate the cell solutions at 37 °C for 1-2 hours (can go up to three, but try to avoid doing it for that long). 17. Plate the cells on plates containing the correct antibiotic. Each transformation requires two plates. Add 500 µL of solution to one plate, spread with the spreading stick, and then spread the spreading stick on the second plate without adding any solution to it. This creates a dilution plate in case you have thousands of colonies on the first plate. It is roughly a 1:100 to 1:200 dilution. 18. Grow the plates up overnight at 37 °C. Do not leave for longer than 24 hours, as contaminants might have a chance to grow and the plates could dry out. If your cuvette arcs (bright flash and loud popping noise during electroporation): 1. Clean the electroporator lid. 2. Wash out and sterile the cuvette with ethanol – the cells have been pretty much killed and will not be usable in plating, so you need to restart. 3. Add less DNA to the cells (reduce by 25%-33%). 4. Add an additional 15 µL of WB buffer to the solution.

b. Colony selection, Stock Streak Plating, and Culture Preparation

1. Carefully decide which cell characteristics you are looking for. 2. Identify colonies with the characteristic that are separated by a small distance from other colonies – this makes it easier to pick only the colony you want 3. With a sterile toothpick or sterile pipette tip, lightly touch down on the colony to pick up cells 4. Keeping the toothpick or tip oriented so that the cells are on the bottom, gently streak the cells on a new plate with the correct antibiotic. DON’T DISCARD THE TIP/TOOTHPICK. The streak should be around 2 cm or so long, and should be separated from other streaks on the new plate. 5. If you want to grow the cells up in a liquid culture (for glycerol stock and DNA prep), you can place the tip/toothpick into a tube with 5-6 mL LB. There should be enough cells still on the tip/toothpick to have a dense culture overnight. If there is a strong chance that not all colonies picked will be correct (see RFP example above), 2-3 of these cultures should be made for each construct; otherwise 1-2 will usually be enough. 6. If colonies are used to grow up liquid culture, label the streak and the culture tube with the same number, so if the colony is incorrect, you know which tube is incorrect. 7. Repeat for as many colonies as you think you will need (15-25 is generally good for streaks) 8. Put the plate into the 37 °C incubator overnight 9. Re-evaluate the streaks for indications that they are not the right colony. Toss any liquid cultures corresponding to incorrect colonies.

c. Environmental safety?

No environmental safety issues arise from our project. As mentioned above, proper laboratory practice is employed and all parts are present within vectors containing antibiotic resistance. These practices along with the fastidious nature of our system limit any environmental threat.

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?

None of the BioBrick parts used raise any safety concerns. We operate under BSL 2 safety regulations, therefore no persons on our team are permitted to handle or manipulate anything of a pathogenic nature.

3. Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?

Yes, Utah State University has an Institutional Biosafety Committee, linked here. We have discussed our project with the committee and they have approved our work. Since our laboratory is used for other synthetic biological research the approval was straightforward.

Utah State University has its own biosafety rules, linked here. The University has rules and regulations regarding the correct disposal and clean-up of biological waste material. All iGEM team members are trained by the Environmental Health and Safety department (EH&S) at Utah State University. This training covers basic laboratory safety procedures and practice. It is required that everyone (including graduate students and staff) who works in a laboratory at Utah State University take this day long course. In addition, all undergraduates were trained specifically on biological safety by experienced graduate advisors and faculty before they were allowed to work in the laboratory. For more information about Utah State University laboratory safety training please see this link. The United States has strict regulations and guidelines for biosafety. The National Institute of Health (NIH) has guidelines for working with recombinant DNA. See: NIH. The centers for disease control and prevention (CDC) also has guidelines for biosafety. See: CDC

4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

The basis of iGEM is to standardize biological part assemble, why don’t we standardize basic safety training as well? Every institute (and country) has different requirements for students to undergo safety training. We believe that a basic online biosafety course (consisting of photos and videos) through iGEM/Registry should be made compulsory for all students willing to participate in iGEM. Once students have successfully completed the online course they should be required to take a quick quiz to see if they learned basic biosafety. Upon completion of the safety course and quiz students would then be eligible to join an iGEM team. Bacteria that are used for iGEM purposes could be encoded with a ‘kill’ gene which prevents the bacteria from growing and reproducing outside of its designated environment.