Team:NTNU Trondheim/Protocols

In this section, the various procedures and techniques we have used during the course of the project are described in detail. Any exceptions from these protocols are given in our lab journal.

Medium
- Preparation of LB-medium
  This is the recipe for the preparation of 1L medium:
  
  1L Water
  
  10g Bactotryptone
  
  5g Yeast extract
  
  5g NaCl
  
  Preparation of LA-medium (1.5%), 15g agar was added to the mix.
  
  After the addition of all the components, the medium was stirred and autoclaved for 20min at 121°C.
- Preparation of LA petri dishes
  0.5mL Antibiotics (CM or AMP)
  
  0.5L LA-medium
  
  Cool the LA-medium for a little while before the addition of the antibiotics. Shake the flask after the addition to get a homogenous solution.
  
  Pour the solution into sterile plates on a sterile bench, and spread the solution even on the plate.
  
  Wait until the agar is set and store the plates in the fridge.
- Preparation of M63B1-medium
  This is the recipe for the preparation of 1L medium:
  
  1L Water
  
  13.6g KH₂PO₄
  
  4.2g KOH
  
  2g (NH₄)₂SO₄
  
  0.15 mg FeSO₄*7H₂0
  
  The pH was adjusted to pH 7.2 with KOH.
  
  After the addition of all the components, the medium was stirred and autoclaved for 20min at 121°C.
  
  1mL 1M MgSO₄*7H₂O was then added to the medium in addition to 1 uL of a stock of b-vitamin (0.9108g b-vitamin in 90 mL). The amount of wanted glucose was also added afterwards to achieve a concentration of 0.4% and 0.8% glucose.
- Preparation of PBS-solution
  This is the recipe for the preparation of 1L solution:
  
  1L Distilled water
  
  8g NaCl
  
  0.2g KCl₂
  
  1.42g Na₂HPO₄
  
  0.24g KH₂PO₄
  
  After adding the substances above, fill up the flask to 90mL with MilliQ (ultrapure water)
  
  The pH was adjusted to 6.5 by adding diluted NaOH (0.2M).
  
  Fill the flask up to 100 mL with more MilliQ and filter sterilize.
- Psi - medium
  20g Tryptone
  
  5g Yeast extract
  
  5g MgCl
  
  Adjust to pH 7.6 with KOH Add water to 1 liter and autoclave
Buffers
- Preparation of TFB1-buffer
  This is the recipe for the preparation of 200mL buffer (use a 250mL flask):
  
  0.588g CH₃CO₂K
  
  2.42g RbCl
  
  0.295g CaCl₂• 2H₂0
  
  1.98g MnCl₂
  
  30mL Glycerol
  
  After adding the substances above, fill up the flask to 180mL with MilliQ (ultrapure water)
  
  The pH was adjusted to 5.8 by adding diluted acetic acid (0.2M).
  
  Fill the flask up to 200 mL with more MilliQ and filter sterilize.
- Preparation of TFB2-buffer
  This is the recipe for the preparation of 100mL buffer (use a 250mL flask):
  
  0.21g MOPS
  
  0.121g RbCl
  
  1.1g CaCl₂
  
  15mL Glycerol
  
  After adding the substances above, fill up the flask to 90mL with MilliQ (ultrapure water)
  
  The pH was adjusted to 6.5 by adding diluted NaOH (0.2M).
  
  Fill the flask up to 100 mL with more MilliQ and filter sterilize.
Making supercompetent E.coli cells
Duration of the protocol: Two days
1. Make a culture of E.coli in a flask with 10mL Psi-medium. Inoculate at the end of the day, and incubate overnight at 37^oC with shaking.
2. Transfer 0.5mL of the culture to a new shaking flask with 100mL Psi-medium. Grow in an incubator with vigorous shaking at 37^oC until A₆₀₀ reaches 0.4-0.5.
3. Place the culture, TFB1 and TFB2 on ice for 5-10 minutes (buffers and pipets is preferred to be cold for the entire protocol).
4. Transfer cells to 50mL sterile chilled polypropylene centrifuge tubes (10 mL in each falcon tube). It is important with clean tubes.
5. Pellet cells at 4^oC and 5.000 x g for 5 minutes. Throw the supernatant away carefully (possibility for a break if the cells stay on ice)
6. Do this step on ice. Gently resuspend the pellet in 0.4 volume of TFB1 (20 mL if the total is 50mL in a falcon tube). Leave it for 15 minutes on ice.
7. Pellet cells for 10 minutes at 2.000 x g and 4^oC. Pour off supernatant carefully.
8. Do this step on ice. Resuspend the pellet in 0.02 volume of TFB2 (carefully).
9. Mark as many 2mL cryo tubes as you will need with strain name and the date.
10. Add 50uL of the resuspended cells into the cryo tubes, and leave them on ice.
11. Flash freeze for 5-10 seconds in liquid nitrogen and store at -80^oC.
Colonypicking to liquid medium
After incubation (agar plates) overnight:
1. Select a single colony. Use an inoculation loop. The loop is sterilized with flame before and after use.
2. Smudge some of the colony with the loop, and transfer the bacteria to a liquid medium.
Miniprep
Plasmid isolation (Miniprep) was performed following the protocol for the ZR Plasmid Miniprep^TM - Classic Kit with slight modification:
1. Transfer 1mL of each cell culture to an eppendorf tube and centrifuge for 20s at 16000g. Discharge the supernatant.
2. Add 200μL resuspension buffer (P1 buffer) to the tube and resuspend the cell pellet by pipetting.
3. Add 200μL lysing buffer (P2 buffer) and mix gently by inverting the tube 4 times. Incubate at room temperature for 2min.
4. Add 400μL neutralization buffer (P3 buffer) and mix carefully until the solution changes colour from pink to yellow. Centrifuge for 2min at 16000g.
5. Place a spin column (Zymo-SpinTM IIN column) in a collection tube and transfer the supernatant from step 4 into the spin column.
6. Centrifuge for 30s at 16000g. Discharge flow-through.
7. Add 200μL Endo-wash buffer to the column and centrifuge for 1min at 16000g.
8. Add 400μL Plasmid-wash buffer to the column and centrifuge for 1 min at 16000g.
9. Transfer the spin column into a clean eppendorf tube. Add 30μL of deionized water directly into the matrix of the column. Incubate for 1min at room temperature.
10. Elute the plasmids in the spin column by centrifuging for 30s in 16000g.
Gel electrophoresis
For 1% agarose gel:
- GelRed or GelGreen (standard 0.8% or 0.4% if separation of smaller fragments)
- TAE or LAB buffer
Preparation of the gel:
1. Pour the GelRed/GelGreen-solution into the gel mould (0.5cm) and let the gel set for 30 minutes.
2. Move the set gel to an electrophoresis chamber. Based on what buffer is used in the GelRed/GelGreen-solution, choose that one for the covering of the gel. Cover the chamber and gel with the chosen buffer.
Restriction digest:
1. DNA (10μL), 10xbuffer (2μL), distilled water (7,5μL) and the restriction enzyme (0,5μL) were mixed in a test tube.
2. The samples were incubated for 1h at 37℃.
3. Loading dye (4μL) were added to each sample
4. A solution of DNA ladder (1kb) was prepared by mixing distilled water (8μL), Gene Ruler (2μL) and loading dye (2μL). Note: 250ng DNA are required per 0.5μL enzymes.
5. DNA ladder (5μL), and the samples with the inserted plasmids (20μL) were added to the wells in the agar gel.
Run the gel:
1. Run the gel at 90V for approximately 30 minutes, and check the migration of fragments.
Gel purification
The purification of DNA was performed following the protocol for the Zymoclean^TM Gel DNA Recovery Kit:
1. Add 3 volumes of ADB buffer to each volume of agarose excised from gel.
2. Incubate the sample for 5-10min at 50℃.
3. Transfer the melted sample to a Zymo-spin^TM column assembled into a collection tube. Centrifuge for 60s at 16000g. Discard the flow-through.
4. Add 200μL DNA Wash Buffer and centrifuge for 30s at 16000g.
5. Repeat step 4.
6. Discard the flow-though collected in the collection tube.
7. Add 20μL deionized water directly onto the matrix of the spin column. Centrifuge for 60s at 16000g.
8. Assemble the spin column into a clean eppendorf tube and transfer the flow-through from the previous step back into the column matrix. Centrifuge for 60s at 16000g.
Transformation of competent cells
1. Thaw competent cells on ice for 10min.
2. Mix 2μL of mini-prepped plasmids with ~50μL competent cells in a tube. Pipette up and down while stirring the pipette tip gently for 5-10s.
3. Incubate the cells on ice for 20min.
4. Heat shock the cells for 45s at 42℃. Immediately put the samples on ice for 2min.
5. Add 900μL LB medium to the cells. Incubate at 37℃, 200rpm for 1h.
6. Spread plate 100μL of the transformation medium into agar plates containing an appropriate selection agent.
7. Incubate the cells at 37℃.
Insertion of anti-luxS gene and amplification of the wanted plasmid with PCR
1. Suspend the reverse anti-luxS primer (26.2nmol) and forward anti-luxS primer (30nmol) with 261μL and 299μL water, respectively, to obtain 100μM solutions. Dilute each primer (10μL) with water (990μL) to obtain a final concentration of 1μM. Insert the 20bp of anti-luxS gene into the wanted plasmid (2) via PCR by the following steps:
2. Add the forward anti-luxS primer (6μL) and the reverse anti-luxS primer (6μL) to a PCR tube.
3. Add pgRNA (2) (0.5μL, 44.2ng/μL) to the PCR tube.
4. Add Takara pre-mix (2x6.5μL) to the solution.
5. Amplify by PCR, and store in the freezer.
Preparation of a 96-well plate and Crystal Violet Assay
Assay used for quantifying the amount of biofilm in a 96-well plate.

Preparation of a 96-well plate:
- Dillute bacterial cultures to 0,1 OD.
- Transfer 100 μL of dilluted bacterial cultures to each well in a 96-well plate.
- Incubate the six plates for 0, 3, 5, 8, 24, and 30 hours at 37 °C.
- Clean each well with 100 uL physiological saline.
- Add 100 μL 100% methanol to each plate
- After 15 minutes remove the supernatant and let the plate dry.
- Add 100 μL 0,1% Crystal Violet solution to each well.
- After 20 minutes wash the excess Crystal Violet away with distilled water.
- Add 150 μL 33% acetic acid to each well.
- Measure absorbance at 590 nm.

Gibson Assembly

The setup for Gibson Assembly was prepared by following after the HiFi DNA Assembly protocol provided by NEB [1]

Gibson Assembly

Set up the following reaction on ice:

	2-3 fragment Assembly	4-6 fragment Assembly
Recommended DNA Molar Ratio	Vector:insert = 1:2	Vector:insert = 1:1
Total Amount of Fragments	0.03 - 0.2 pmols = X uL	0.2 - 0.5 pmols = X uL
NEBuilder HiFi DNA Assembly Master Mix	10 uL	10 uL
Deionized H₂O	10-X uL	10-X uL
Total Volume	20 uL	20 uL

Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.

[1] https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf

Site-Directed Mutagenesis
Procedure
- Prepare a PCR mix by mixing 7.5 pmol designed primers (FWD and REV), >100 ng template (we used >50 ng), and 12.5 μL Takara HiFi pre-mix.
- Introduce site directed mutagenesis by PCR.
XTT Assay
Procedure
- Remove supernatant.
- Wash with 100 μL physiological saline.
- Add 150 μL PBS.
- Add 50 μL XTT reagent.
- Incubate for 4 hours at 37 °C.
- Shake plate for 5 minutes and measure Abs480.

Ligation

The setup for ligation of DNA fragments was prepared by following after the Ligation Protocol with T4 DNA Ligase, provided by NEB [1].

Procedure

1. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.)

Component	20 μL reaction
T4 DNA Ligase Buffer (10X)* 2 μL	2 μL
Vector	The volume is calculated according to its size and concentration
Insert	The volume is calculated according to its size and concentration
Nuclease-free water	To 20 μL
T4 DNA Ligase	1 μL

* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

Gently mix the reaction by pipetting up and down and microfuge briefly.
For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
Heat inactivate at 65°C for 10 minutes.
Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

References:

https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

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Team:NTNU Trondheim/Protocols

Preparation of a 96-well plate:

Crystal Violet Assay:

Gibson Assembly

Procedure

Procedure

Procedure