Preparation of Basic Experimental Material

1. LB

Materials:

Tryptone
Yeast extract
NaCl AR
dH₂O
Conical flask
Microwave
Magnetic stirrer and rotor

Protocol:

Weigh reagents in the following proportions:

Substance	Concentration
Tryptone	10 g/L
Yeast extract	5 g/L
NaCl	10 g/L

Deposit them all into the flask.
Add distilled water to the expected volume.
Stir the mix until the powders are completely dissolved.
Autoclave for 20 minutes at 121°C.
Store at 4°C.

2. LB Agar

Materials:

Tryptone
Yeast extract
NaCl AR
Agar
dH₂O
Conical flask
Microwave
Magnetic stirrer and rotor

Protocol:

Weigh reagents in the following proportions:

Substance	Concentration
Tryptone	10 g/L
Yeast extract	5 g/L
NaCl	10 g/L

Deposit them all into the flask.
Add distilled water to the expected volume.
Stir the mix until the powders are completely dissolved.
Add 1.5% weight agar into the flask.
Autoclave for 20 minutes at 121°C.
Store at 4°C.

3. Preparation of LB-Antibiotics

If the medium does not contain agar: Add one thousandth of a volume of antibiotic solution, mix thoroughly.
If the medium does contain agar: When the temperature is reduced to approximately 45°C, add one thousandth of a volume of antibiotic solution, then mix it thoroughly. After antibiotics are added, it should not be heated again.

4. Preparation of Agarose Gel

Materials:

Agarose
1x TAE
Graduated cylinder
Conical flask
Pipettor and pipette
Microwave
10,000x nucleic acid dye

Protocol:

Add the exact weight of agarose to the measuring cylinder to ensure the correct concentration.
Add 1x TAE of the required volume.
Heat the solution in the microwave until it boils.
Take the cylinder out and let it cool down until it can be touched by hands.
Add nucleic acid dye that is 1/10,000 of the total volume of the system.
Pour into electrophoresis box, place the comb, and wait until it solidifies.
Take out the comb carefully.

5. TY Medium

Materials:

Tryptone
Yeast extract
CaCl₂ AR
dH₂O
Conical flask
Microwave
Magnetic stirrer and rotor

Protocol:

Substance	Concentration
Tryptone	5 g/L
Yeast extract	3 g/L
CaCl₂	0.6 g/L

Deposit them all into the flask.
Add distilled water to the expected volume.
Stir the mix until the powders are completely dissolved.
Autoclave for 20 minutes at 121°C.
Store at 4°C.

6. TY Agar

Materials:

Tryptone
Yeast extract
CaCl₂ AR
Agar
dH₂O
Conical flask
Microwave
Magnetic stirrer and rotor

Protocol:

Substance	Concentration
Tryptone	5 g/L
Yeast extract	3 g/L
CaCl₂	0.6 g/L

Deposit them all into the flask.
Add distilled water to the expected volume.
Stir the mix until the powders are completely dissolved.
Add 2% weight agar into the flask.
Autoclave for 20 minutes at 121°C.
Store at 4°C.

If needed, 5% sucrose can be added to TY or TY agar.

Molecular Biology Experiment

1. Genome Extraction

Materials:

Centrifuge tubes (1.5 mL & 2.0 mL)
G+ Bacteria Genomic DNA Kit (Zomanbio)
RNase A (10 mg/mL) (Zomanbio)
ddH₂O
Ethanol
Centrifugal machine
Vortex machine
Water bath

Protocol:

Centrifugate 1-5 mL bacteria solution for 1 min at 13,400 ×g to retain the pellet.
Add 500 μl cell suspension solution (with Lysozyme added) to the bacterial pellet, vortex until the pellet is re-suspended. Incubate at 37℃ for 30 minutes, inverting every 5-10 minutes. Centrifuge at 13,400 ×g for 2 minutes and thoroughly remove the supernatant.
Add 225 μl Buffer A and mix thoroughly.
Add 6 μl RNase A (10 mg/mL) solution, vortex for 5 seconds and mix well.
Add 25 μl lysis solution S and mix thoroughly. Incubate at 56℃ for 20 minutes, inverting 1-2 times during this period.
Add 250 μl Buffer B, vortex for 5 seconds and mix thoroughly.
Add 250 μl ethanol, vortex for 15 seconds. Transfer the mixture to the column and place the column in the collection tube.
Centrifuge at 13,400 ×g for 30 seconds, discard the supernatant. Place the absorption column in the collection tube.
Add 500 μl Buffer C, centrifuge at 13,400 ×g for 30 seconds, discard the supernatant, and place the column back into the collection tube.
Add 600 μl Washing Solution W2 (with ethanol added), centrifuge at 13,400 ×g for 30 seconds, discard the supernatant, and place the column back into the collection tube.
Add an additional 500 μl Washing Solution W2 (with ethanol added), centrifuge at 13,400 ×g for 30 seconds, discard the supernatant, and place the column back into the collection tube.
Centrifuge for 2 minutes at 13,400 ×g. Open the column and let it sit at room temperature for a few minutes.
Place the column in a new 1.5 mL centrifuge tube, add 100-200 μl elution buffer TE, and let it sit at room temperature for 2-5 minutes.
Centrifuge for 2 minutes at 13,400 ×g. Transfer the liquid in the collection tube to the column.
Dispose of the column and store the eluate at -20℃.

2. PCR

2.1 General PCR

Goal: Amplify a gene or add homology arms to a sequence.

Materials:

PCR tubes (200 μl)
GoldenStar T6 Super PCR Mix (1.1x)
Forward primer (10 μM)
Reverse primer (10 μM)
Template
Thermocycler

Protocol:

Add in a PCR tube:

Substance	Volume (system 25μl/50μl)
Golden Mix	(23-x) μl/(46-x) μl
Forward primer (10 μM)	1 μl/2 μl
Reverse primer (10 μM)	1 μl/2 μl
Template	x μl (depends)

Place the tube in the thermocycler and start corresponding program.
After the sample temperature decreases to 4℃, store the result at -20℃.

2.2 Fusion PCR

Goal: Connect two DNA fragments containing homologous arms together.

Materials:

PCR tubes (200 μl)
GoldenStar T6 Super PCR Mix (1.1x)
Template (containing homologous arms)
Forward primer (10 μM)
Reverse primer (10 μM)
Thermocycler

Protocol:

Add in a PCR tube:

Substance	Volume (system 25μl/50μl)
Golden Mix	(23-x-y) μl/(46-x-y) μl
Template1	x μl (depends)
Template2	y μl (depends)

Add 1 μl/2 μl of each of the two primers.
Place the tube in the thermocycler and start corresponding program.
After the sample temperature decreases to 4℃, store the result at -20℃.

2.3 Colony PCR

Goal: To determine whether a certain sequence exists in the thallus.

Materials:

PCR tubes (200 μl)
2 x Taq Master Mix (dye) / 2 x T5 Super PCR Mix (Basic)
Forward primer (10 μM)
Reverse primer (10 μM)
ddH₂O
Thermocycler

Protocol:

Add in a PCR tube:

Substance	Volume (system 25μl)
2 x Taq Master Mix / 2 x T5 Super	12.5 μl
Forward primer (10 μM)	1 μl
Reverse primer (10 μM)	1 μl
ddH₂O	10.5 μl

Usually, we configure more premix than the required number of systems in a 1.5 mL centrifuge tube, then separate them into PCR tubes.
Place the tube in the thermocycler and start corresponding program.
After the sample temperature decreases to 4℃, stop the program and use the results for AGE detection.

3. Plasmid Extraction

3.1 Zomanbio

Materials:

Centrifuge tubes (1.5 mL & 2.0 mL)
Fast Plasmid Miniprep Kit (Zomanbio)
Centrifugal machine
Vortex machine

Protocol:

Centrifugate 1-5 mL bacteria solution 1 min at 13,400 x g, retain precipitation.
Add 250 μl Buffer K1 (RNase added) in the bacteria precipitation, vortex it until the thallus is resuspended.
Add 250 μl buffer K2, gently mix it until the liquid looks clear.
Add 350 μl buffer K3, immediately mix it gently.
Centrifugate the tube 2 min at 13,400 ×g, transfer the supernatant to column and put it in the collection tube, centrifugate 30-60 s at 13,400 ×g. Discard supernatant, replace the column into the collection tube.
If the bacteria is E. coli BL21 (DE3) or Pseudomonas fluorescens 2P24, add 500 μl Deproteinized solution W1 in column and centrifugate 30-60 s at 13,400 ×g. If the bacteria is E. coli DH5 α, skip this step.
Add 500-600 μl Washing Solution W2 (ethanol added) in column and centrifugate 20 s at 13,400 ×g. Repeat this step.
Centrifugate 1 min at 13,400 ×g.
Put the column in a new centrifuge tube, add 80-150 μl elution buffer TE. Place it for 1 min.
Centrifugate 1 min at 13,400 ×g. Discard the column and store the result at -20℃.

3.2 TransGen

Materials:

Centrifuge tubes (1.5 mL & 2.0 mL)
EasyPure Plasmid Miniprep Kit (TransGen)
Centrifugal machine
Vortex machine

Protocol:

Before use, RNase A was added to RB and stored at 2-8℃. Add different volumes of 100% ethanol to WB.

LB Media Volume	RB (μl)	LB (μl)	NB (μl)
5 mL	250	250	350
5-10 mL	500	500	700
10-15 mL	750	750	1,050
15-20 mL	1,000	1,000	1,400

Take the overnight culture, centrifuge at 10,000 ×g for 1 minute, and remove the supernatant.
According to the table, add colorless solution RB (containing RNase A), vortex to resuspend the bacterial pellet, ensuring no small clumps remain.
According to the table, add blue solution LB, gently invert 4-6 times to mix, ensuring complete lysis of the bacteria, forming a blue and clear solution. The color change from semi-transparent to clear blue indicates complete lysis.
According to the table, add yellow solution NB, gently mix 5-6 times (the color change from blue to yellow indicates complete mixing and neutralization), until a compact yellow precipitate forms, then let it stand at room temperature for 2 min.
Centrifuge at 12,000 ×g for 5 min, carefully transfer the supernatant to a centrifuge column. Centrifuge at 12,000 ×g for 1 min, discard the flow-through.
Add 650 μl solution WB, centrifuge at 12,000 ×g for 1 min, and discard the flow-through.
Centrifuge at 12,000 ×g for 1-2 min to thoroughly remove residual WB.
Place the centrifuge column in a clean centrifuge tube, add 20 μl EB to the center of the column, and let it stand at room temperature for 1 min. (Pre-warming EB in a 60-70°C water bath improves the outcome).
Centrifuge at 10,000 ×g for 1 min to elute the DNA, and store the eluted DNA at -20°C.

3.3 Mei5bio

Materials:

Centrifuge tubes (1.5 mL & 2.0 mL)
M5 HiPer Multi-color Plasmid Miniprep Kit (Mei5bio)
Centrifugal machine
Vortex machine

Protocol:

Column Equilibration: Add 400 μL of Buffer BL to the centrifugal adsorption column, let it stand for 1 minute, centrifuge at 12,000 rpm at room temperature for 1 minute, discard the waste liquid in the collection tube, and place the centrifugal adsorption column back into the collection tube.
Take 1-5 mL of overnight culture, centrifuge at 12,000 rpm at room temperature for 1 minute, and remove the supernatant as completely as possible.
Add 250 μL of Solution I, vortex or pipette thoroughly to resuspend the bacteria evenly, resulting in a uniformly turbid brown-red color.
Add 250 μL of Solution II, gently invert to mix well, allowing the bacteria to fully lyse until the solution becomes clear and viscous purple-red.
Add 350 μL of Solution III, immediately and gently invert to mix, a precipitate with red and yellow layers will be visible, continue mixing until it turns completely yellow, let it stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 5 min.
Carefully transfer the supernatant to the centrifugal adsorption column, let it stand for 2 min to allow the plasmid to bind fully with the silica membrane of the column. Centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate in the collection tube.
Add 500 μL of Buffer WB2, centrifuge at 12,000 rpm at room temperature for 30 seconds, and discard the filtrate in the collection tube. Repeat this step once.
Centrifuge at 12,000 rpm at room temperature for 2 min to dry the residual liquid.
Place the centrifugal adsorption column in a new 1.5 mL plastic centrifuge tube (provided by the user), add 50-100 μL of elution buffer EB, let it stand at room temperature for 2 min.
Centrifuge at 12,000 rpm for 1 minute, and the solution at the bottom of the centrifuge tube is the plasmid DNA. Store the eluted DNA at -20℃.

4. Plasmid Digestion

Goal: To cleave a vector using restriction enzymes to prepare them for ligation.

Materials:

rCutSmart Buffer (10x)
ddH₂O
200 μL centrifuge tube
Water bath at 37℃
Water bath at 65℃

Protocol:

Substance	Volume
Enzymes	0.1 μl
Enzymes	0.1 μl
Plasmid	x μl (depends)
rCutSmart Buffer (10x)	1 μl
ddH₂O	(8.8-x) μl

Keep warm in a water bath at 37℃ for 1 hour.
Keep warm in a water bath at 65℃ for 20 minutes.
Correct bands were extracted after electrophoresis.

Materials:

10X QuickCut Green Buffer
ddH₂O
200 μL centrifuge tube
Water bath at 37℃
Water bath at 80℃

Protocol:

Substance	Volume
Enzymes	1 μl
Plasmid	x μl (depends)
10X QuickCut Green Buffer	1 μl
ddH₂O	(8-x) μl

Keep warm in a water bath at 37℃ for 1 hour.
Keep warm in a water bath at 80℃ for 15 minutes.
Correct bands were extracted after electrophoresis.

5. Agarose Gel Electrophoresis (AGE)

Goal: Separating DNA bands according to their length, and estimating the length and DNA concentration of each band. When digested products or recombinant products are subjected to electrophoresis, care should be taken to use the original plasmids for control.

Materials:

Ready-made agarose gel
DNA sample
10 x loading buffer (dye)
DNA Ladder/Marker
1 x TAE
Electrophoresis tank

Protocol:

Place the gel on the gel tray within the electrophoresis system, make sure the wells are located at the negative electrode.
Mix loading buffer with the DNA sample if the sample is not coloured. Usually, the loading buffer has one tenth the volume of the sample, but the volume should not be less than 0.3 μl.
Load either the DNA ladder or the samples within the wells. Make sure that the sample or ladder sinks to the bottom of the well. The volume should be between 3 μl to 5 μl, depending on the well’ s capacity.
Run the gel for 20-40 minutes, depending on the situation. Ensure the voltage is set to 100-150 V.

6. DNA Extraction from Agarose Gel

Materials:

Centrifuge tubes (1.5 mL)
EasyPure Quick Gel Extraction Kit (TransGen)
ddH₂O
Centrifugal machine
Vortex machine
Water bath

Protocol:

Carefully cut the target band on agarose gel and put it in a centrifuge tube.
Add 600 μl GSB. Heat it at 55℃ using a water bath for 6-10 minutes. Mix intermittently to ensure the gel is completely melted.
After the gel dissolves, allow the solution to cool to room temperature, transfer the liquid to a column, and put it in the collection tube. Centrifuge for 60 seconds at 10,000 ×g. Discard the flow-through liquid.
Add 650 μl WB, centrifuge for 60 seconds at 10,000 ×g. Discard the flow-through liquid.
Centrifuge for 1-2 minutes at 10,000 ×g to completely remove residual WB.
Place the centrifuge column in a clean centrifuge tube, open the cap and let it stand for 1 minute to allow the residual ethanol to evaporate completely. Then add 20 μl of deionized water (pre-warmed) to the center of the column and let it stand at room temperature for 1 minute.
Centrifuge for 60 seconds at 10,000 ×g. Discard the column and store the result at -20℃.

7. Gibson Assembly

Goal: To assemble the different DNA parts retrieved by PCR.

Materials:

PCR tubes (200 μl)
ClonExpress Ultra One Step Cloning Kit
ddH₂O
Thermocycler

Protocol:

Calculate the required amount of DNA for the recombination reaction according to the formula:
- Single-fragment: Optimal cloning vector usage = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol) Optimal insert fragment usage = [0.04 x number of base pairs of insert fragment] ng (0.06 pmol)
- Multi-fragment: Optimal cloning vector usage = [0.02 x number of base pairs of cloning vector] ng (0.03 pmol) Optimal usage for each fragment = [0.02 x number of base pairs of each fragment] ng (0.03 pmol)
Prepare the following reaction system on ice:

Components	Recombination Reaction	Negative Control-1	Negative Control-2	Positive Control
Linearized Vector	X μl	X μl	0 μl	1 μl
n Insert Fragments	Y1+Y2…+Yn μl	0 μl	Y1+Y2…+Yn μl	1 μl
2x ClonExpress Mix	5 μl	0 μl	0 μl	5 μl
ddH₂O	to 10 μl	to 10 μl	to 10 μl	to 10 μl

Gently mix well with a pipette (do not vortex mix), briefly centrifuge to collect the reaction liquid at the bottom of the tube.
Single-fragment recombination reaction, 50°C, 5 min; cool to 4°C or immediately place on ice.
Multi-fragment recombination reaction, 50°C, 15 min; cool to 4°C or immediately place on ice.

Microbiology Experiment

1. Transformation of chemically competent E. coli

Materials:

LB medium
LB agar plates with antibiotics (depends)
Crushed ice
Plasmid used to transform
Chemically competent cell
Centrifuge tube (1.5 mL)

Protocol:

Add 100 μl chemically competent cell and 10 μl of plasmid in the tube, mix gently and softly.
Incubate the tube for 30 minutes on ice.
Incubate for 90 seconds at 42°C, then immediately place on ice for 2 minutes.
Add 700-900 μl (depends) of LB liquid medium.
Incubate for 45 minutes under agitation at 200 rpm.
Centrifuge for 5 minutes at 6,000 ×g.
Remove 950 μl of supernatant and resuspend the bacteria with 50 μl of this supernatant.
Spread the solution on a LB agar plate containing the relevant antibiotic.
Incubate in incubator at 37°C overnight.
Check for colonies the next day.

2. General bacterial concentration testing

Materials:

Nano spectrophotometer
Test solution

Protocol:

Turn on the Nano spectrophotometer and select the measurement mode for bacterial suspension.
Calibrate the spectrophotometer with standard samples to ensure accuracy.
Add the bacterial suspension to a cuvette and place it in the spectrophotometer to measure the absorbance. Ensure cleanliness and optical path alignment.
Record the concentration of the bacterial suspension.

3. Bacterial plating

3.1 Dilution coated plate method

Materials:

Bacteria solution cultured for a period of time
Solid medium
Spreader

Protocol:

Add some bacteria solution onto the surface of the medium.
Spread from one side of the plate, draw a few circles, and then overlap the streaks. Use a sterilized spreader and ensure it’ s not too hot.
Place the plates in the incubator with clean surfaces. Temperature varies by bacteria type.

3.2 Plate streaking

Materials:

Bacteria solution cultured for a period of time
Solid medium
Inoculating loop

Protocol:

Dip inoculating loop into some bacterial liquid.
Streak on one side of the plate, do a quarter turn, and then overlap the streaks.
Place the plates in the incubator with clean surfaces. Temperature varies by bacteria type.

4. Phenotype inducing

Induced by IPTG

Culture the bacteria solution for 12 hours, ensuring OD₆₀₀ is no less than 0.8.
Add 16 μl of 50 mg/ml IPTG.
After induction, culture at 37°C.
Sometimes, a non-induced control group is used.

Agarose Gel Electrophoresis Analysis

Gel Preparation (1.5%)
Electrophoresis: 3-5 μl of sample, 5 μl of marker, constant voltage of 150V for 30-60 minutes.

Methods

1. Magnetic Bead-based Protein Purification

1.1. Buffers Preparation

All purification buffers were prepared with ultrapure water and filtered through 0.22 μm or 0.45 μm membranes prior to use. Two buffer systems were employed for soluble and inclusion body proteins, respectively.

For Soluble His-Tagged Proteins:

Binding/Wash Buffer: 50 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, pH 8.0.
Elution Buffer: 50 mM Tris-HCl, 300 mM NaCl, 300 mM Imidazole, pH 8.0.

For His-Tagged Proteins from Inclusion Bodies:

Binding/Wash Buffer: 50 mM Tris-HCl, 300 mM NaCl, 8 M Urea, pH 8.0.
Elution Buffer: 50 mM Tris-HCl, 300 mM NaCl, 8 M Urea, 300 mM Imidazole, pH 8.0.

1.2. Sample Preparation

Bacterial/Yeast Expressed Proteins:
Cell pellets were resuspended in ice-cold Binding Buffer (1:10-20, w/v) supplemented with 1 mM PMSF, 1% Triton™ X-100, and 1 mM TCEP. Cells were lysed by sonication on ice. The lysate was clarified by centrifugation, and the supernatant was collected for purification.

Secreted Proteins from Yeast, Insect, or Mammalian Cells:
Culture supernatant was collected by centrifugation. If necessary, the supernatant was dialyzed against Binding Buffer to remove interfering substances (e.g., EDTA, imidazole, reducing agents) or concentrated via ammonium sulfate precipitation followed by dialysis.

1.3. Magnetic Beads Equilibration

Ni-IDA Magnetic Beads suspension was thoroughly resuspended. An appropriate volume (e.g., 0.2-0.5 mL of 20% suspension per 2 mg target protein) was transferred to a tube. The beads were immobilized using a magnetic separator, and the storage solution was removed. The beads were then washed three times with an equal volume of Binding Buffer.

Protocol:

Binding: The clarified sample was incubated with the equilibrated beads for 1 hour at 4°C with gentle mixing.
Washing: The beads were immobilized, and the flow-through was collected. The beads were washed three times with 1 mL of Wash Buffer.
Elution: Bound proteins were eluted by incubating the beads with 1 mL of Elution Buffer for 5 minutes. This step was repeated 2-3 times to maximize recovery.
Concentration and Analysis: The eluate was concentrated using a 30 kDa molecular weight cut-off centrifugal filter unit. Protein purity and yield were analyzed by SDS-PAGE.
Regeneration: Beads were sequentially washed with: 50 mM Tris-HCl (pH 8.0) containing 6 M Guanidine-HCl, deionized water, 0.1% SDS, deionized water, 70% ethanol, deionized water, 100 mM EDTA (pH 8.0), deionized water, and finally recharged with 100 mM NiSO₄ overnight at 15°C with shaking. Regenerated beads were stored in 20% ethanol at 4°C.

2. Bacteriophage Adsorption and DNA Injection Assay by Transmission Electron Microscopy (TEM)

2.1. Phage-Bacteria Co-incubation

Lactococcus lactis subsp. cremoris 3107 (wild-type and mutant strains) were grown in GM17 medium to an OD_620nm of 0.25 (early exponential phase). Bacteriophage TP901-1 lysates were dialyzed against SM Buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO₄, pH 7.5) and adjusted to a titer of approximately 1×10⁸ PFU/mL.
A mixture containing 0.15 mL bacterial culture, 0.15 mL phage lysate, and 0.2 mL of a 1:10 dilution of GM17 broth in quarter-strength Ringer’ s solution was incubated at 25°C for 10 minutes.

2.2. Sample Processing for TEM

The reaction mixture was immediately placed on ice and centrifuged at 12,000 × g for 10 minutes at 4°C. The pellet was washed twice with ice-cold quarter-strength Ringer’ s solution and finally resuspended in 50 μL of the same solution.
Carbon films floated on mica plates were directly applied to the bacteria-phage mixtures for 10 minutes to adsorb the complexes. The specimens were then negatively stained with 1% uranyl acetate (aqueous solution) for a few seconds.
Micrographs were acquired using either an EM 300 or a Tecnai 10 transmission electron microscope (Philips) operating at an acceleration voltage of 80 kV.

3. Bacteriophage Concentration and Titration

3.1. Phage Concentration by PEG Precipitation

A 200 mL culture of the host bacterium (Salmonella strain G33T-2) was grown in LB broth at 37°C with shaking (220 rpm) to an OD₆₀₀ of ~0.6. Purified phage (250 μL) was added, and the culture was incubated overnight. Bacterial debris was removed by centrifugation (10,000 × g, 20 min, 4°C) and filtration through a 0.22 μm membrane. DNase I and RNase A were added to the filtrate and incubated at room temperature for 30 minutes.
The phage particles were precipitated by adding NaCl to a final concentration of 1 M, followed by PEG-8000 to 10% (w/v), with stirring. The solution was incubated on ice for at least 1 hour and then centrifuged at 11,000 × g for 20 minutes at 4°C. The pellet was resuspended in 1.6 mL SM Buffer. Residual PEG and contaminants were removed by repeated chloroform extraction (3-5 cycles) until the aqueous phase was clear. The concentrated phage titer was determined and stored at 4°C.

3.2. Phage Spot Titer Assay

A fresh colony of E. coli MG1655 was inoculated into 5 mL LB medium and grown to the exponential phase at 37°C with shaking.
The phage lysate was serially diluted (up to 10^-9) in sterile LB broth. A 10 μL aliquot of each dilution was spotted onto a lawn of the host bacterium prepared in a double-layer agar assay (bottom layer: 1.5% agar-LB; top layer: 0.7% agar-LB mixed with 1 mL of bacterial culture).
Spots were allowed to dry, and plates were incubated at 37°C for 8-12 hours. Plaques were counted to determine the phage titer in Plaque-Forming Units per mL (PFU/mL).