Team:Waterloo/Experiments

Experiments

Inoculation

1. Aseptically pipette 5 mL sterile liquid broth into a test tube.
2. Add appropriate antibiotic, if required, to desired concentration using a pipette. Volumes depend on the specific antibiotic but there is a list adhered to the top door of the -20℃ freezer in the iGEM lab.
3. When pipetting the antibiotic, tilt the test tube so that the broth is close enough to the top of the tube that you can add the antibiotic to the broth. Dropping it in won’t work because you will typically be adding approximately 2.5 μL and the drop will be too small to fall.
4. If inoculating from frozen stock, work quickly! This is very important because if the cells thaw completely they will have a shorter lifetime in the freezer. Always keep frozen stocks on ice.
5. Take an autoclaved, and thus sterile, wooden inoculation stick and scrape some frozen stock from the tube and dip the stick into the liquid media and stir around.
6. Discard inoculation stick into another container, not the waste! Inoculation sticks are reusable because they can be autoclaved multiple times.
7. If inoculating from a plate, use a sterile wooden inoculation stick and touch a colony on the plate, then dip it into the liquid media and stir it around. Again, do not the discard sticks.
8. Place the tubes in a 37℃ incubator on the shaker for 12-24 hours. If possible, angle the tubes because it improves oxygenation of the culture as it shakes.

Nanodrop

The NanoDrop (ND-1000) is a small-scale spectrophotometer that can detect DNA concentration and give an estimate of sample purity.

1. Open the nanodrop application on the computer and login to the iGEM account using the password “bio”.
2. Click on “Nucleic acid” then begin to initialize the unit.
3. Put some MilliQ water on a KimWipe and wipe down the pedestal and sampling arm.
4. Add 1.0 μL of ddH2O, then 1.0μL of elution buffer to blank.
5. Load 1.0 μL of sample and click “Measure” to determine the concentration and absorbance ratios. Record these below. Between trials, wipe the machine with a KimWipe. Make sure to leave machine clean and how you found it.

Polymerase Chain Reaction (PCR)

1. Per reaction: In a 200 μL tube, mix:
   • 1 μL Forward Primer (10 μM)
   • 1 μL: ReversePrimer (10 μM)
   • 10 μL 2X Taq/Q5/Phusion Master Mix
   • 8uL Sterile, nuclease-free water

2. Add template individually to each sample. Template may be a few cells (i.e. picked colony, small volume of liquid culture), or extracted/purified DNA (i.e. plasmid, genome, linear). Typically, add 1 μL of ~1ng/uL extracted/purified DNA (dilute in nuclease-free water if needed) or 1 μL of overnight culture. For colony PCR, simply touch the colony with a sterile tip and mix that into your reaction tube.

3. Place samples in thermocycler.

   	Q5		Taq		Phusion
   Step	Temperature (in C)	Time (min:sec)	Temperature (in C)	Time (min:sec)	Temperature (in C)	Time (min:sec)
   Initial denaturation	98	0:30	95	0:30	98	0:30
   Denaturation	98	0:05 - 0:10	95	0:15 - 0:30	98	0:05 - 0:10
   Annealing	58-72	0:10 - 0:30	45-68	0:15 - 1:00	45-72	0:10 - 0:30
   Extension	72	0:20 - 0:30 per kb	68	1:00 per kb	72	0:15 - 0:30 per kb
   Final extension	72	2:00	68	5:00	72	5:00 - 10:00
   Hold	4	Infinity	4	Infinity	4	Infinity

   • Annealing temperature depends on the primers you’re using.

   • Note that this is for standard PCR (where the template is extracted DNA). If your template is DNA in whole cells (i.e. colony PCR), then initial denaturation should be 5-10 min to lyse the cells.

Plasmid miniprep

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps in the solution; it should have uniform turbidity.
3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
5. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.
6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifuge for 1 minute. Discarding the flow-through is optional.
8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
10. Add ≥ 30 μl DNA Elution Buffer to the centre of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Gel electrophoresis

Setting up your rig:

1. Usually we use the small rig with the corresponding small tray (holds a 50 mL agarose gel).
2. Tape the edges of the tray. This needs to be water-tight, as you will set your gel here, so then the liquid solution can solidify.

Choosing a comb: There are several reasons you might want to run a gel, which correspond to different choices of comb.

1. If you are running a gel to visualize a result, use the comb with the smallest teeth, and use the side with the thinnest teeth. Usually you run a small amount of sample in a gel like this, so the wells don’t need to be large.
2. If you are running a gel for the purposes of gel extraction, use the comb with the largest teeth, and use the side with the thickest teeth. Usually you run a large amount of sample in a gel like this, so the wells need to be large.

Pouring and running your gel:

1. Add agarose into a flask to make the concentration of agarose 0.8-1.2% (e.g. 0.5 g agarose to make 50 mL 1% agarose gel)
2. Add desired amount of 1X TAE running buffer (50 mL for small tray, but use more if you are running a larger gel).
3. Microwave for 30 seconds. Using rubber mitt, vigorously swirl the partially-dissolved agarose in the flask.
4. Microwave for 15 seconds. Using rubber mitt, vigorously swirl the dissolved agarose in the flask. Visually inspect the solution to make sure there is no undissolved agarose particles floating in it. Keep microwaving (in short 15 second intervals or so) until fully dissolved.
5. Add Gel Red at 1:10 000 concentration (50 mL gel needs 5 μL gel red).
6. Pour gel into tray.
7. Put comb into place.
8. Let it sit for 20 minutes. Wait until gel loses some transparency and looks more “white” and cloudy.
9. Once the gel has set, remove the tape from the rig and replace the tray in the rig so the ends of the tray are open.
10. Add additional 1X TAE running buffer to the rig. Buffer should cover the top of the gel.
11. Carefully remove comb from gel.
12. Load samples and appropriate DNA ladder (generally a 1kb ladder is used).
13. Attach the lid to the gel rig and plug the lid into the electrophoresis machine.

Gel extraction

1. Weigh microfuge tube.
2. Excise the DNA fragment to be purified from the agarose gel using a razor blade, scalpel or other clean cutting tool. Use care to trim excess agarose. Transfer it to a 1.5 ml microcentrifuge tube and weigh the gel slice.

3. Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the slice (1mg of gel : 4uL of buffer).

   • Note:
     • Calculation: weight of gel piece extracted = (weigth of gel in microfuge tube) - (weight of microfuge tube)
     • If the volume of the dissolved sample exceeds 800 μl, the loading of the sample onto the column should be performed in multiple rounds to not exceed the volume constraints of the spin column.
4. Incubate the sample between 37–55°C (typically 50°C), vortexing periodically until the gel slice is completely dissolved (generally 5–10 minutes).
5. Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
6. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
7. Repeat wash (Step 6).
8. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If in doubt, re-spin for 1 minute before placing into clean microfuge tube.
9. Add ≥ 6 μl of DNA Elution Buffer to the centre of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA.

DNA Purification

1. Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample volume of 20–100 μl is recommended.

   Sample type	Ratio of binding buffer : sample
   dsDNA > 2 kb (plasmids, gDNA)	2:1
   dsDNA < 2 kb (some amplicons, fragments)	5:1
   ssDNA (cDNA, M13)	7:1

2. Insert column into collection tube and load sample onto column. Spin for 1 minute, then discard flow-through.
3. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discard flow-through.
4. Repeat wash (Step 3).
5. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to next step.
6. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Heat shock transformation

1. Acquire ice and enough competent cells for your experimental samples and controls.
2. Thaw competent cells on ice.
3. Get new 1.5 ml tubes, and label them with your different sample names. One tube will be a positive control (e.g. uncut vector alone) to test if your transformation technique worked.
4. Add 50 μL of competent cells to each labelled tube and then add your DNA. If DNA is from a ligation, add the entire volume into the tubes. If it is from a miniprep, usually add 1-10 μL depending on the DNA concentration.
5. Incubate on ice for 30 minutes.
6. Heat shock at 42℃ for 45 seconds and then immediately put the tubes back onto ice.
7. Incubate on ice for 5 minutes.
8. Add 750 μL of LB broth to each tube then incubate at 37℃ for 45 minutes - 1 hour with shaking.
9. After incubation you will plate the cells onto selective media so only transformants will grow.

Frozen stock preparation

1. Prepare an overnight culture of the cells that are to be made into frozen stock.
2. Aliquot 1 mL of overnight culture into a 1.5 mL microfuge tube.
3. Pellet cells by spinning for 1 minute at 16,000 x g. Discard supernatant.
4. Resuspend in 1 mL fresh liquid broth.
5. In a 1.5 mL cryovial tube, add 150 μL of stock glycerol solution.
6. To that cryovial tube, add 850 μL of freshly resuspended cells (from step 4).
7. Pipette up and down to mix.
8. Label cryovial tube appropriately and update the strain list.
9. Store in -80°C freezer.

Competent cell preparation

1. Inoculate 99 mL of LB with 1000 μL of overnight culture and grow at 37°C to an OD of 0.300-0.400 (such as 0.368).
2. Place cells on ice once they reached an OD of 0.300-0.400 (such as 0.368) and chill for 20-30 minutes (swirling periodically for even cooling).
3. After 20 minutes, split culture into 2 cold Falcon tubes. There should be 50 mL in each then be centrifuged at 4°C for 15 minutes 13000g.
4. Decant LB and resuspend each in 10 mL cold MgCl2.
5. Centrifuge for 15 minutes at 4°C at 13000 x g.
6. Decant and re-suspend cells in each Falcon tube in 10 mL of ice cold CaCl2 and allow to sit on ice for 20 minutes of incubation.
7. Centrifuge for 15 minutes at 4°C and 2000 x g (this was done twice because the pellet was not fully formed).
8. Decant and re-suspend in 5 mL cold CaCl2 + 15% glycerol.
9. Aliquot l00-200 μL into prechilled sterile 1.5mL microfuge tubes on ice.
10. Flash freeze tubes using liquid nitrogen and place in -80°C freezer.

Methionine sharing

1. Prepare an overnight culture of methionine non-producers and optogenetic methionine producers in the turbidostat.
2. Spin down 650 µL of the optogenetic population at 13000 RPM for 1 minute.
3. Remove the supernatant.
4. Resuspend cells in 25 mL of M9 media that lacks methionine (M9-met).
5. Spin down at 13000 RPM for 1 minute.
6. Aspirate the media.
7. Resuspend in 25 mL of M9-met with 25 µL of appropriate antibiotic for your population’s resistance.
8. Grow until OD nears 0.4.
9. Set aside 50 µL of the optogenetic population.
10. Spin down the rest at 13000 RPM for 2 minutes.
11. Aseptically filter sterilize the supernatant.
12. Take 100 µL of methionine non-producer culture, spin down 13000 RPM for 1 minute.
13. Draw off supernatant.
14. Resuspend in 1 mL of M9-met.
15. Set up experimental tubes and controls according to the following table.

Test Tube #</		Control</		Components</
1	Experimental	→ M9 - met with CcaS/R v2.0 JT2 removed (3 mL) → Empty JT2 (50 µL)
2	Positive	→ M9 - met with CcaS/R v2.0 JT2 removed (3 mL) → CcaS/R v2.0 JT2 (50 µL)
3	Positive	→ M9 - met with CcaS/R v2.0 JT2 removed (3 mL) → Empty JT2 (50 µL) → Met (150 µL)
4	Negative	→ M9 - met with CcaS/R v2.0 JT2 removed (3 mL)
5	Negative	→ Stock M9 - met (3 mL) → Met (150 µL)
6	Negative	→ Stock M9 - met (3 mL) → Empty JT2 (50 µL)
7	Positive	→ Stock M9 - met (3 mL) → Empty JT2 (50 µL) → Met (150 µL)

16. Grow overnight at 37C
17. Record OD readings

Growth Curves

1. Inoculating cells

• Inoculate cells into LB medium with appropriate antibiotics for selection
• The next morning re-inoculate from the overnight culture into M9 media with the appropriate antibiotics

2. Adjusting Cells to Light

• Once the cells have reached an OD of > 0.6 re-inoculate 100ul of culture into 35mL of M9 drop out media (-methionine)
• Place cells into the turbidostat with a stir bar
• Incubate at 35 degrees with stirring under green light for 3h
• NOTE: The intensity of green light should be the same intensity you want to use for the growth curve

3. Growth Rate Measurement

• After 3h of adjusting the cells to the green light begin taking OD 600 measurements every 20 min until the cells reach an OD of ~0.8
• Plot the ln(OD 600 values) vs Time
• Calculate the growth rate by taking the slope of the linear portion of the curve

Waterloo iGEM 2018

© 2018 Waterloo iGEM