Basic Molecular Biology Techniques:

PCR using Q5 Hi-Fi DNA polymerase Master Mix

Reaction setup:

The template DNA should keep an amount of 1-1000 ng

Thermocycler conditions

Clarification:

1. The sequences in red are where the primers anneal with the biobrick affixes. And the 5’overhangs side with the sequences in red are designed for Gibson Assembly with linearized pNZ8148 digested by PstI due to a 21-nucleotide overlap between them.
2. The reason of setting two different cycles is that, at the beginning of PCR, the annealed parts between primers and templates are only sequences in red. As PCR proceeds, 5’overhangs are added to the entire genes, so the rest reaction of PCR becomes the one between the whole primers and the whole genes, because of which the Tm varies compared to the former.

Applications in this project: Amplification of BBa_K2309028, BBa_K2309003 and BBa_K2309004

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PCR purification using QIAquick PCR purification kit

1. Add 500 μl Qiagen buffer PB
2. Spin through a column twice, discard flow-through
3. Wash 1x with 700 μl buffer PB
4. Wash 2x with 760 μl buffer PE
5. Discard liquid, spin dry at 17000g for 3 min
6. Elute into a new tube twice with 50 μl of TE (100 μl total)

(Can be also referred to from iGEM protocol)

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Gel extraction

Reagents:

1.5 ml micro-centrifuge tube; 2 ml collection tube; a QIAquick spin column; Isopropanol (100%); Buffer QG; Buffer PE; Buffer EB

Procedure:

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100 μl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
4. Add 1 gel volume isopropanol to the sample and mix.
5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 μl, load and spin/apply vacuum again.
6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
7. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow through and place the QIAquick column back into the same tube.

Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt- ended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.

8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.

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Gibson Assembly

Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases）

Applications in our project: Assembly of BBa_K2309004, BBa_K2309005, BBa_K2309022 and BBa_K2309028 with pNZ8148 respectively.

Procedure:

1. Prepare an ice box, put every material on ice.
2. Add 0.02-0.5 pmols DNA fragment (which has been amplified by PCR using primer 1 and primer 2—see at PCR protocol) into PCR tube. (The mass of DNA can be calculated by NEB-calculator)
3. Add 1/3 amount of DNA inserts PstI-digested pNZ8148 plasmid in the tube.
4. Add 2X Gibson Assembly Master Mix (produced by NEB) to the tube.
5. Fill the tube with dH_{2O to a total volume of 20 μl.}
6. Incubate the samples in a thermocycler at 50℃ for 15 minutes. (can be longer) Store samples on ice or at -20℃ for subsequent examination or Transformation.

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Digestion (NEB enzymes)

• Single-enzyme digestion

Reagents:



Usually the volume of reaction can vary:



Procedure:

1. Add 100-1000 ng DNA into an empty 1.5 ml microcentrifuge tube.
2. Add 5μl 10×NEB buffer (dependent on enzymes) to the tube.
3. Complement ddH₂O to a total volume of 49 μl system.
4. At last, add 1μl restriction enzyme and incubate the reaction system at proper temperature (normally 37 ℃) for 1 hour. The digestion efficiency can be increased through a prolonged incubation time.

(The work conditions of corresponding restriction enzyme can be referenced on NEB official website)

Application in this project: pNZ8148 plasmid digestion by PstI

• Double-enzyme digest

Incubate at 37℃ for 1 hour. Heat kill at 80℃ for 20 minutes.

(0.5 μl BSA can be added in reaction to increase digest efficiency, and buffer should be decided depending on the enzymes used to maximize the cutting efficiency)

Applications in this project: PstI and EcoRI for Linearized pSB1C3, PstI and EcoRI for BBa_2309022, BBa_2309023 and BBa_2309027.

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Ligation

Procedure:

1. Add 2μl digested plasmid backbone (25ng).
2. Add equimolar digested fragment.
3. Add 1μl T4 DNA ligase buffer.
4. Add 0.5μl T4 DNA ligase.
5. Add ddH₂O to 10ul volume.
6. Ligate at 16℃ for 30 minutes. Heat kill at 80℃ for 20 minutes.
7. Transform 1-2μl of the product.

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Transformation of plasmids in E.coli DH5ɑ

(Details can be referenced at iGEM official protocol)

Applications in this project: Cloning of BBa_K515005

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Isolation of plasmid in E.coli DH5ɑ

Reagents:

AxyPrep Plasmid Miniprep Kit (Miniprep column; 2 ml Microfuge tube; 1.5 ml Microfuge tube; Resuspension Buffer S1 (contains RNase A); Lysis Buffer S2; Neutralization Buffer S3; Wash buffer Buffer W1; Desalting Buffer W2; Eluent Buffer (2.5 mM Tris-HCl))

Procedure:

1. Collect 4 ml of overnight LB culture. Centrifuge at 12,000×g for 1 minute to pellet the E. coli. Decant or pipette off as much of the supernatant as possible.
2. Re-suspend the bacterial pellet in 250 μl of Buffer S1 by vortex. Please be sure that the bacteria are completely re-suspended before proceeding.
3. Add 250 µl of Buffer S2, and mix by gently inverting the tube 4-6 times.
4. Add 350 µl of Buffer S3, and mix by gently inverting 6-8 times. Centrifuge at 12,000×g for 10 minutes to clarify the lysate.
5. Insert the required AxyPrep Plasmid Miniprep column into the fittings on the 2 ml Microfuge tube. Transfer the clarified supernatant from Step 4 into the Miniprep column.
6. Add 500 µl of Buffer W1 to Miniprep column. Centrifuge at 12,000×g for 1 minute.
7. Pipette 700 µl of Buffer W2 along the wall of the column to remove residual salt. Centrifuge at 12,000×g for 1 minute. Repeat this wash step with a second 700 µl aliquot of Buffer W2.
8. Centrifuge at 12,000×g for 1 minute to purge residual Buffer W2 from the binding membrane.
9. Transfer the Miniprep column into a clean 1.5 ml Microfuge tube. To elute the purified plasmid DNA, add 60~80 µl of Eluent buffer to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 minute.

Application in this project : pSB1C3-BBa_K515005 extraction from DH5ɑ transformant

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Preparation of competent Lactococcus lactis

Materials:

1. 0.5 M sucrose, 10% glycerol (store at 4℃)
2. 0.5 M sucrose, 10 glycerol, 50 mM EDTA (store at 4℃)

Procedure:

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Electroporation for L. lactis

Materials:

Procedure:

1. Place 40 μl competent cells in a pre-chilled electroporation cuvette with 1 μl DNA (reconstituted in TE-buffer) and keep the cuvette on ice.
2. Use the Electroporator with following adjustments:
   2500 V (12.5 KV/cm)
   25 μF
   200 Ω - Pulse (normal reading is 4.5-5 msec)
3. Add 1 ml G/L-M17B + 20 mM MgCl₂ + 2 mM CaCl₂.
4. Keep the cuvette for 5 min on ice and incubate 1-1.5 h at 30 °C.
5. Plate 10 µl, 100 µl, 900 µl on M17agar with glucose or lactose and antibiotics (depends on plasmid) - Incubate 1-2 days at 30 °C.

Tips:

1. Lactococcus lactis grows very slowly in G/L-SGM17B. Leaving out the sucrose is possible but can lower the transformation efficiency. The medium for cell recovery must contain MgCl₂ and CaCl₂.
2. The sterilization temperature can be set at 108 degrees Celsius in case of carbonization.

Applications in our project: Transformation of BBa_K2309005, BBa_K2309004 and BBa_K2309028 (independent in pNZ8148)

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Plasmid DNA isolation from Lactococcus lactis

Materials:

Procedure:

1. Use 5 ml full grown culture
2. Spin down 10 min 3000 x g or more (up to 6000 x g)
3. Re-suspend pellet in 250 μl THMS-buffer + 2 mg/ml lysozyme in Eppendorf cup
4. Incubate 10 min at 37 °C
5. Add 500 μl 0.2 N NaOH + 1% SDS, shake carefully (no vortex)
6. Incubate 5 min on ice
7. Add 375 μl ice-cold 3 M potassium acetate pH 5.5, shake carefully
8. Incubate 5 min on ice
9. Spin 5 min in Eppendorf centrifuge (Maximum Rotation Speed)
10. Take out supernatant and add to 2 ml new Eppendorf cup
11. Add 0.7-1 ml 2-propanol
12. Incubate 5-10 min at room temperature
13. Spin 10 min in Eppendorf centrifuge (Maximum Rotation Speed)
14. Wash pellet carefully with 70% ethanol
15. Dry the pellet (vacuum)
16. Dissolve the pellet in 50 μl TE or sterile water

(For our project, the plasmid we used is pNZ8148, which is chloramphenicol-resistant)

Applications in our project: pNZ8148 extraction

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Dot blot

Materials:

1. TBS: 20 mM Tris-HCl; 150 mM NaCl; pH 7.5
2. TBS-T: 0.05% Tween20 in TBS
3. 1. BSA/TBS-T: 0.1% BSA in TBS-T

Procedure:

1. Have nitrocellulose membrane ready, draw grid by pencil to indicate the region which are going to blot.
2. Using narrow-mouth pipette tip, spot 2 μl of samples onto the nitrocellulose membrane at the center of the grid.Minimize the area that the solution penetrates (usually 3-4 mm diam.) by applying it slowly.
3. Let the membrane dry.
4. Block non-specific sites by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). Use 10cm Petri Dish for reaction chamber.
5. Incubate with primary antibody (0.1-10 μg/ml for purified antibody, 1:1000 to 1:100000 dilution for antisera, 1:100 to 1:10000 for hybridoma supernatant) dissolved in BSA/TBS-T for 60 min at RT.
6. Wash three times with TBS-T (3 x 5 min).
7. Incubate with secondary antibody conjugated with HRP (for optimum dilution, follow the manufacturer’s recommendation) for 60 min at RT.
8. Wash three times with TBS-T (15 min x 1, 5 min x 2), then once with TBS (5 min).
9. Incubate with ECL reagent for 1 min, cover with Saran-wrap (remove excessive solution from the surface), and expose X-ray film in the dark room. Try several different lengths of exposure.
10. Compare the signal from your unknown sample to that of standard and estimate the concentration.

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Anti-microbial peptides assay:

Pre-experiment: CFU measurement

Reagents:

Staphylococcus aureus solution (37℃, 200rpm, overnight culture); LB agar plates

Procedure:

1. Dilute Staphylococcus aureus solution with x10, x100, x500, x1000 concentration gradients. Vortex each concentration of solutions for 1-3min;
2. Use microscope to count the cell number of different concentration gradient.
3. Culture each concentration gradient of Staphylococcus aureus solution on LB agar. Add 20μl of solution on plate for each concentration. Do 3 repeats for each gradient.
4. Overnight culture at 37℃ condition.
5. After overnight culture, count colonies.

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Inhibition ring

Reagents:

Staphylococcus aureus solution (37℃, 200rpm, overnight culture); Anti-microbial peptides stock solutions (1mg/ml) (or AMPs: LL-37, Grammistin-Pp1, GF-17) (China Peptides, Suzhou); LB agar plates

Procedure:

1. Defreeze AMP solutions at 4℃.
2. Add 170μl of LL-37, Grammistin-Pp1, GF-17 solutions to form a 1:1:1 LL-37+Grammistin-Pp1+GF-17 solution.
3. Measure OD₆₀₀ value of Staphylococcus aureus after 9 hours’ culture.
4. Inoculate 100μl diluted S. aureus (OD₆₀₀≈0.4) on each medium.
5. Pipette 20μl each peptide solution on each paper disc (LL-37, Grammistin-Pp1, GF-17, and LL-37+Grammistin-Pp1+GF-17).
6. Dry all discs.
7. Place 2 paper discs with peptide, 1 paper disc with positive control (chloramphenicol) and 1 negative control (buffer: PBS+BSA+HAC) for each plate.
8. Overnight culture at 37℃ condition.

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AMPs efficiency and MIC assay

Reagents:

1. Staphylococcus aureus solution (37℃, 200rpm, overnight culture);
2. Anti-microbial peptides stock solutions (1mg/ml) (or AMPs: LL-37, Grammistin-Pp1, GF-17) (China Peptides, Suzhou); LB agar plates

Procedure:

• AMPs dilution
1. Take AMPs stock solution from -20℃ freezer.
2. Dilute AMPs by PB buffer. The conc. gradients of diluted AMPs as follow:
   LL-37: 80ug/ml, 40ug/ml, 20ug/ml, 10ug/ml, 5ug/ml, 1ug/ml, 0.5ug/ml;
   Grammistin-Pp1: 40ug/ml, 20ug/ml, 10ug/ml, 6ug/ml, 3ug/ml, 1ug/ml, 0.5ug/ml
   GF-17: 20ug/ml, 10ug/ml, 6ug/ml, 3ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml
3. After each dilution, vortex 1-3min for each of concentration of AMPs
• Staphylococcus aureus dilution
1. Measure OD₆₀₀ value of original Staphylococcus aureus solution.
2. Dilute Staphylococcus aureus solution 500 times and 1000 times respectively in different EP tube by PB buffer;
3. After dilution by PB buffer, vortex each conc. gradients of solution 1-3min.
• AMPs and Staphylococcus aureus interaction
1. Pipette 50μl diluted AMPs solution and 50μl diluted Staphylococcus aureus solution (1/500 and 1/1000 diluted) into one EP tube.
2. Vortex each EP tube for 1-3 min.
3. Incubate 3.5 hours at 37℃, 180rpm condition.
4. Plate 30μl mixed solution into the LB agar medium. Overnight culture at 37℃ condition.

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Biofilm formation test

Reagents:

1. Staphylococcus aureus solution (37℃, 200rpm, overnight culture)
2. Anti-microbial peptides stock solutions (1mg/ml) (or AMPs: LL-37, Grammistin-Pp1, GF-17) (China Peptides, Suzhou)
3. LB Broth
4. 96-well transparent plate

Procedure:

1. Dilute 200μl Staphylococcus aureus of stationary phase to 2ml volume using LB.
2. Mix 100 μl diluted S. aureus (in step 1) and 100 μl peptide solutions of different concentrations and transfer the mixture in 96-well plate.
3. Add 200μl LB to each well in column 1 of both plates.
4. Add 20μl SA (from step 2) and 180μl LB to each well in column 2 of both plates.
5. Add 200μl SA+AMP solution (from step 3) to wells 1-4, 5-8 in column 3,4,5,6,7,8,9,10,11,12 (4 repeats for each concentration of AMP).
6. Seal plates with tape and incubate at 37℃ in plate reader for 24h.

Our setting for LL-37 and Grammistin-Pp1:

Sample plate for GF-17 and Mixed anti-microbial peptides:

Biofilm staining

1. Pipette LB out of the 96-well plate.
2. Add 200ul ddH₂O in each well.
3. Pipette H₂O out.
4. Add 200μl ddH₂O in each well.
5. Pipette H₂O out.
6. Incubate for 15min in 37 ℃.
7. Air dry (for around 15min).
8. Add 200μl crystal violet (0.1%) in each well.
9. Wash with ddH₂O three times.
10. Add 200μl 95% ethanol.

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Growth curve measurement

Reagents:

1. Staphylococcus aureus solution (37℃, 200rpm, overnight culture);
2. Anti-microbial peptides stock solutions (1mg/ml) (or AMPs: LL-37, Grammistin-Pp1, GF-17) (China peptides, Suzhou)
3. LB Broth.

Procedure:

1. Add 100ml LB Broth into each 250ml conical flask (5 in total);
2. Label each of conical flask as LL-37, Grammistin-Pp1, GF-17, Mix, and Staphylococcus aureus.
3. Add 300μl Staphylococcus aureus solution (OD₆₀₀≈4) into each conical flask.
4. Culture at 37℃, 180rpm.
5. Measure OD₆₀₀ value after each hour, when OD₆₀₀ value is 0.05, add 300μl anti-microbial peptides into each conical flask except the one labeled Staphylococcus aureus (the concentration of peptides is 1mg/ml). Measure OD₆₀₀ value every 2 hour until the Staphylococcus aureus grow into the stationary phase (12 hours)

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Nisin induction of gene expression in Lactococcus lactis:

Testing of S. aureus sensing pathway

(Transformant with pNZ8148 harboring BBa_K2309005):

1. Grow 5 ml successfully transformed bacteria culture overnight, 30 °C.
2. Dilute 1/25 in 2 x 10 ml fresh medium (30 °C). Grow until the OD₆₀₀ ≈ 0.4.
3. Induce one 10 ml culture with 0.5 - 5 ng/ml nisin (typically 1 ng/ml is used) and keep the other 10 ml culture as negative control.
4. Incubate 2 - 3 hours/
1. Aliquot both control group and experiment group into 96-well plate.
2. Test the fluorescence under the set of 485 nm excitation and 510 nm emission.

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Autolysis test

(Transformant with pNZ8148 harboring BBa_2309004):

1. Grow 5 ml successfully transformed bacteria culture overnight, 30 °C.
2. Dilute 5-fold with fresh medium.
3. Aliquot 200μl culture into each well of 96-well plate. Grow at 30℃ in plate reader with a regular test every 30 minutes until stationary phase (OD₆₀₀≥3).
4. Induce with 0.5-5 ng/ml nisin (usually 1 ng/ml). Continue to observe the growth curve with a regular OD₆₀₀ test every 1 hour.