Introduction
We first created standard protocols for experiments that are likely to be repeated multiple times, allowing us to efficiently replace the relevant values on the day of the experiment. Additionally, we identified areas for improvement in some protocols and optimized them accordingly each time. (For more details, please refer to the https://2024.igem.wiki/botchanlab-tokyo/engineering.)
If you are interested in the protocols for individual assays or detailed daily experimental protocols, please refer to the Results Pages and Lab Notebook
We hope this will assist teams with less experience, like ours.
Standard Protocols
Preparation of LB Medium[2][3][4]
Procedure Overview
1. Mix 6 g of tryptone, 3 g of yeast extract, 3 g of NaCl, and 600 mL of distilled water, adjusting the pH to 7.0 using small amounts of aqueous HCl or NaOH if necessary.
2. Pour the solution into a medium bottle, loosened the lid slightly, and placed it in a basket designed for autoclaving.
3. Pour water into the autoclave to a level where the bottom of the rack was slightly submerged, placed the basket inside, set the lid, and tightened the handle securely. After confirming that the steam valve was properly closed, we started the autoclave cycle at 121°C for 20 minutes (in reality, it takes about an hour to complete the autoclave process, including the time for pressure and temperature to build up).
4. After the set time passed, the temperature and pressure inside the autoclave decreased, and the exhaust valve opened at around 70°C. We removed the bottle when it had cooled enough to hold by hand.
5. Once the solution had cooled sufficiently, we tightened the lid of the bottle and stored it at 4°C.
Escherichia Coli Nissle 1917 (EcN) Cultivation
Procedure Overview
1. Dispense 5 ml of LB liquid medium into shaker culture tubes.
2. Using the tip of a pipetman, collect colonies from a plate and add to the LB liquid medium.
3. Incubate at 37℃ with shaking at 225 rpm.
Note: EcN was provided by the National Institute of Infectious Diseases.
Preparation of Competent Cells[1]
Day 1: Pre-Culture
In a clean bench, add LB medium to a 200 mL Erlenmeyer flask, cover it with a lid, and sterilize it in an autoclave. The conditions are 121°C, 2 atm, for 20 minutes. After autoclaving, wait until it cools down to a temperature that can be handled, and wipe off any moisture. Keep the sterilized flask covered with aluminum foil until it is used the next morning.
Before using the clean bench, switch off the UV light, turn on the fan, and disinfect the items to be used (two colony tubes, LB medium, pipettes, tips, tube rack) with ethanol before bringing them into the clean bench. All subsequent procedures are performed inside the clean bench.
Add 3 mL of LB medium to the colony tubes, pick a colony from the EcN plate with a pipette tip, add it to the medium, and suspend it. Label the inoculated colony on the plate with a circle and write the date. Close the culture tube loosely, labeling it with “EcN,” the date, and the initials of the experimenter. Place the culture tubes in the shaker, and incubate overnight at 37°C and 225 rpm. The culture tubes should be placed at a 45-degree angle and fixed in the shaking direction. Confirm the shaker is set to 37°C before leaving.
Day 2: Main Culture
Confirm that the pre-culture appears cloudy, turn off the shaker, and take out the tubes. Set the shaker to 18°C for the main culture and cool it down. Add 1 mL of the pre-cultured E. coli to the 200 mL of LB medium prepared the previous day, and shake it at 18°C and 120 rpm for 16 hours. Prepare liquid nitrogen and transport it to the lab for use on the next day.
Day 3: Preparation of Competent Cells
In the clean bench, take 3 mL of the bacterial suspension, and measure the OD660 with LB medium as a blank. If the OD660 exceeds 0.8, dilute the culture as needed. Prepare an ice box with ice and dissolve ITB (ice-cold solution).
Measure the OD660 of the culture every 45 minutes. Once it reaches 0.55~0.60, stop the culture and immerse the flask in ice water for about 10 minutes. Transfer 35 mL of the culture to 50 mL conical tubes, and centrifuge at 2500g at 4°C for 10 minutes to form a pellet. Decant the supernatant, and completely remove any remaining liquid with a pipette. Resuspend the pellet gently with 80 mL of ice-cold ITB and centrifuge again under the same conditions.
After centrifugation, decant the supernatant and remove any remaining liquid with a pipette. Resuspend the pellet in 20 mL of ice-cold ITB. Add 1.5 mL of DMSO (final concentration 7%), mix by shaking, and leave on ice for 10 minutes. Meanwhile, prepare liquid nitrogen, pouring it into a container. After the boiling stops, add more liquid nitrogen.
Dispense the suspension into 1.5 mL tubes in appropriate amounts and quickly freeze them in liquid nitrogen. Perform this step as quickly as possible. Once frozen, transfer the tubes to an ice box and store them in a -80°C freezer.
*Note: The transformation efficiency of the competent cells should be confirmed at a later date.
Transformation by Heat Shock Method[5]
Heat Shock
1. Label the tube containing competent EcN cells (100 µL) and immerse it in ice.
2. Add the plasmid DNA solution gently to the tube with the competent cells and lightly tap the tube to mix.
3. Leave the tube on ice for 30 minutes.
4. Quickly transfer the tube to a 42°C heat block and incubate for 45 seconds (strict timing).
5. Immediately return the tube to ice for 2 minutes.
6. Add 900 µL of pre-warmed SOC medium (37°C) and incubate the mixture at 37°C for 60 minutes with shaking (225 rpm). Add enough SOC medium to bring the total volume to 1 mL.
Plating
7. After the recovery incubation, transfer 1 mL of the cell suspension to a 1.5 mL tube and centrifuge at 5000g for 5 minutes to pellet the cells.
8. Discard the supernatant and resuspend the pellet in 100 µL of LB medium.
9. Spread 100 µL of the resuspended cells on an LB plate.
10. If a large number of colonies is expected, such as when using a high plasmid concentration, perform an appropriate dilution.
11. Label the plate with the necessary information and incubate it overnight at 37°C, inverted.
Miniprep (NucleoSpin® Plasmid EasyPure)[6]
1. Centrifuge 2-10 mL of E. coli culture medium at 12,000*g for 30 seconds and remove as much of the supernatant as possible.
2. Add Buffer A1 (+ RNase A) 1,150 μL to the cell pellet and dissolve completely by vortexing or pipetting.
3. Add 250 μL of Buffer A2 and mix gently by inverting the tube 5 times. Do not vortex.
4. Allow to stand at room temperature for 2 minutes or until the lysate has clarified.
5. Add 350 μL of Buffer A3 and mix gently by inverting the tubes until the blue color of the LyseControl turns completely colorless. Do not vortex.
6. Centrifuge the solution of 5. at 12,000*g for 3 minutes at room temperature.
7. Set a NucleoSpin Plasmid EasyPure Column in a Collection Tube (2 ml).
8. Add up to 750 μL of the supernatant from 6. to the column and centrifuge at 1,000 to 2,000*g for 30 seconds.
9. Discard the filtrate and place the column in the same collection tube.
10. Add 450 μL of Buffer AQ to the column and centrifuge at 12,000*g for 1 minute.
11. Carefully remove the column from the Collection Tube so that the tip of the column does not touch the filtrate.
12. Set the column in a micro tube (1.5 ml: prepare by yourself).
13. Add 50 μL of Buffer AE, incubate at room temperature for 1 minute, and centrifuge at 12,000*g for 1 minute. (Composition of Buffer AE: 5 mM Tris-HCl, pH 8.5)
14. Repeat 12.13. once more.
15. Store the eluate as plasmid solution at -20°C.
PCR[7][8]
Prepare the PCR tubes according to the table below (place the following reagents in the 0.2 mL volume tubes in descending order of volume, with the enzymes placed last).
| Component | 50 µL reaction | Final concentration |
|---|---|---|
| Nucleas-Free Water | 20 µL | up to 50 µL |
| 1 ng/µL template | 1 µL | 1 ng |
| 50 µM Foward Primer | 2 µL | 0.2~0.3 µM (10~15 pmol) |
| 50 µM Reverse Primer | 2 µL | 0.2~0.3 µM (10~15 pmol) |
| TaKaRa ExPremier DNA Polymerase (2x) | 25 µL | 1X |
| total | 50 µL |
Run PCR under the following conditions.
| STEP | TEMP | time |
|---|---|---|
| 1 | 94℃ | 60 sec. |
| 2 | 98℃ | 10 sec. |
| 3 | X℃ | 15 sec. |
| 4 | 68℃ | 30 sec./kb |
| 5 | repeats 2~4 | 30 cycles |
| 6 | 20℃ |
Creation of Agarose Gel
1. Add 1 g of Agarose S (Nippon Gene) to 100 mL of TAE buffer in an Erlenmeyer flask and heat in a microwave until the solution becomes clear.
2. Add 3 µL of Midori Green Extra, mix well by swirling, and pour the solution into a gel cassette with a comb. Cover it with aluminum foil and let it sit until it solidifies.
3. Once solidified, remove the comb and carefully take out the agarose gel. Wrap the gel in plastic wrap, then cover it with aluminum foil. Store the prepared agarose gel in a cold room.
DNA Electrophoresis[9]
1. Mix 10 µL of sample with 1.1 µL of 10x Loading Buffer.
2. Place the gel on the electrophoresis system.
3. Pour TAE buffer up to 2 mm above the gel.
4. Apply 5.0 µL of Quick-Load purple 1 kb plus DNA Ladder to lane 1.
5. The samples were sequentially loaded in the lanes, starting from the second lane.
6. Electrophoresis at 100 V for 30 min.
7. Visualise bands with iBright1500.
Gel Extraction and PCR Clean-up[10]
1. Add 200 µL of Buffer NTI per 100 mg of agarose.
2. Incubate at 55℃ for 20 min.
3. Vortex the sample gently every 2-3 minutes until the gel is completely dissolved.
4. Continue incubating after the gel has dissolved completely.
5. Check that the solution is yellow and proceed to the next step.
6. Place the NucleoSpin Gel and PCR Clean-up Column into the Collection Tube (2 mL).
7. Add solution 1 to the column and centrifuge at 11,000 x g for 30 s.
8. Discard the filtrate and place the column in the same Collection Tube.
9. Add 700 µL of Wash Buffer NT3 to the column and centrifuge at 11,000 x g for 30 seconds.
10. Discard the filtrate and place the column in the same Collection Tube.
11. Repeat steps 8 and 9 once more (2nd wash).
12. Centrifuge the column at 11,000 x g for 1 minute.
13. Set the column in a microtube.
14. Add 30 µL of Buffer NE, incubate at room temperature for 1 min and centrifuge at 11,000 x g for 1 min.
15. Return 30 µL of the extracted solution to the column once more and repeat steps 12,13.
16. Collect 1 µL of the purified solution and measure the concentration, 260/280, 260/230 on a NanoDrop2000.
NEBuilder Assembly[11][12]
1. Enter the base pairs (bp) and concentration of the DNA fragments to be used into the NEBuilder Calculator and calculate the optimal amount to be added.
2. Incubate the prepared sample at 50°C.
Incubation time:
・For3 or fewer fragments: 15 minutes.
・For 4 or more fragments: 60 minutes.
References
[1] (Japanese)Inoue Method for Competent Cell Creation. Retrieved July 1, 2024.
[2] (Japanese)Sigma-Aldrich. "Microbial Media." Retrieved July 1, 2024.
[3] Sonnenborn, Ulrich, and Jürgen Schulze. The Non-Pathogenic *Escherichia Coli* Strain Nissle 1917 – Features of a Versatile Probiotic. Microbial Ecology in Health and Disease. 2009 Dec 26; 21 (3–4): 122–58. doi:10.3109/08910600903444267.
[4] DSMZ. Search. Retrieved July 1, 2024.
[5] University of Zurich. 2022 iGEM Team. Retrieved July 1, 2024.
[6] Macherey-Nagel. NucleoSpin Plasmid Easypure Instruction Manual. Retrieved September 3, 2024.
[7] (Japanese)Takara Bio. TaKaRa Ex Premier™ DNA Polymerase. Retrieved August 4, 2024.
[8] New England Biolabs. Tm Calculator. Retrieved August 4, 2024.
[9] N-Genetics. NE-MWD1P. Retrieved August 4, 2024.
[10] (Japanese)Takara Bio. NucleoSpin® Gel and PCR Clean-up. Retrieved August 4, 2024.
[11] (Japanese)New England Biolabs. NEBuilder® HiFi DNA Assembly Cloning Kit. Retrieved August 10, 2024.
[12] New England Biolabs. NEBuilder HiFi DNA Assembly Reaction Protocol. Retrieved August 10, 2024.