LB Broth

• 10 g Tryptone
• 5g yeast extract
• 10g NaCl
• 1000 mL ddH2O

LB agar

• 15g Agar
• 25g LB Broth
• 1000 mL ddH2O

MacConkey (MAC) Agar medium

• 55g MAC Agar
• 1000 mL ddH2O

7%NaCl TSA medium

• 40g TSA
• 70g NaCl
• 1000 mL ddH2O

Buffer A

• NaCl 29.25g
• Tris 2.42g
• water 1L

Buffer B(pH=8.0)

• NaCl 29.25g
• Tris 2.42g
• water 1L
• imidazole 34.04g

PBS

• Dilute 10×PBS to 1×PBS

PCR

Material:

• KOD OneTM PCR master Mix
• upstream and downstream primers
• plasmid template
• ddH2O

Sequences of primers

Plasmid

Plasmid	Figure	Plasmid	Figure
pET28a-TFP		pET28a-GFP-TFP
pET28a-gp15		pET28a-GFP-gp15

Protocol:

• PCR system:5μl of KOD-one enzyme, 0.3 μl of the upstream and downstream primers, 0.3 μl of the template, and up to 10 μl with ddH2O
• Procedure for PCR is shown below.

Detection of PCR product

Material:

• Agarose
• TAE
• nucleic acid dye

Protocol:

1. Preparation of electrophoresis gel

   Adding 0.5g agarose, 50mL TAE, 2μl of nucleic acid dye and mix.

   Heat, then pour it into mold to solidify.

2. Electrophoresis

   Loading the prepared agarose gel and putting it into the electrophoresis tank.

   Adding 3μl of each PCR product and 3μl marker to the wells of the electrophoresis gel respectively.

   Electrophoresis (120V) for 20 min.

   Comparing the bands with marker to make sure PCR products correspond to the length of plasmid.

Purify the product

The kit used in the experiment : FastPure Gel DNA Extraction Mini Kit (Vazyme)

Material:

• Buffer GDP
• buffer GW
• ddH2O

Protocol:

1. Centrifugate the PCR product in a small centrifuge, and transfer the product to 1.5mL centrifugal tube, then supplement to 100μl with ddH2O.
2. Add 500μl buffer GDP to the mixture.
3. Install the adsorption column into the collection tube, pour the mixture into the adsorption column, and centrifuge it at 10,000rpm for 30s.
4. Pour out the filtrate, put the adsorption column into the collection tube, add 700μl buffer GW (including ethanol) to the adsorption column, centrifuge at 12,000rpm for 30s, and repeat for once.
5. Pour out the filtrate and place the adsorption column into the collection tube then centrifuge at 12,000rpm for 2min.
6. Put the adsorption column into a 1.5mL centrifuge tube, add 20μl ddH2O, after 2min, centrifuge at 12,000rpm for 1min, then discard the adsorption column, save the DNA at -20 °C in the centrifuge tube.

Gibson assembly

Material:

• 2×SeamLess cloning Master Mix
• gene fragment
• linearized plasmid fragment
• ddH2O

Protocol:

1. Add 5μl of 2×SeamLess cloning Master Mix ,300ng of gene fragment, 100ng of linearized plasmid fragment, and up to 10μl using ddH2O.
2. React at 50℃ for 30 min

Preparation of competent cells

Material:

• DH5α strains
• LB broth
• LB agar
• 0.1 mol/L CaCl2 solution

Protocol:

1. Take the DH5α strain from -80°C refrigerator. Absorb a small amount of the bacterial protection liquid and add to LB Broth, paste it on LB agar, then preserve it in 37 ℃ incubator overnight.
2. pick up moist and smooth E.coli single colony from LB agar, add it to 5 mL LB Broth(without antibiotics), and incubate overnight at 37℃ until late log growth. Add 2mL suspension to 100mL LB Broth. Put it in the shaking table and incubate at 37℃ for 2-3h.
3. Transfer the bacterial solution to a cold centrifuge tube on ice for 10-30min, then centrifuge at 4℃, 4000rpm for 10min.
4. Discard the supernatant and reverse it for 1 min to drain the solution
5. Suspend the cells with 10 mL of 0.1 mol/L cold CaCl2 solution, fully mix, and place it on ice. After 30 min , centrifuge at 4℃, 4,000 rpm for 10min.
6. Discard the supernatant and reverse it for 1 min to drain the medium.
7. Add 4 mL of 0.1 mol/L cold CaCl2 solution containing 15% glycerol (sterilized), suspend the cells and place it on ice for a few minutes. Then store it at-80℃.

Transfer the plasmid to DH5α（competent)

Material:

• DH5α competent cells
• LB Broth

Protocol:

1. Take E. coli DH5α competent cells from the -80 °C refrigerator and quickly insert it into the ice box to dissolve it. Add the DNA sample and gently mix.
2. After 20min, use 42°C water bath to heat it for 90 seconds, then quickly put it back to the ice.
3. Add 700μl LB Broth(without antibiotics) to the solution and evenly mix.
4. Put it into shaker and incubate it for 45min (37°C，220rpm), and paste 100μl on the LB agar containing antibiotics. Then preserve it for one night.

Identification of the recombinant plasmids

Material:

• LB Broth
• 2 × Phanta Flash Master Mix (Dye Plus)
• primers

Protocol:

1. PCR: Pick up a single colony from the LB agar, repeatedly blow it into a 1.5 mL centrifuge tube containing 1 mL LB liquid medium, then pick up another colony and blow it into PCR tube containing 10μl PCR reaction system. The 1.5mL centrifuge tube was incubated in a 37℃ 220 rpm shaker for 5 h; the PCR tubes were used for reaction amplification. The PCR reaction system and the reaction procedure are listed below.

Reacion system

Component	Volume
2 × Phanta Flash Master Mix (Dye Plus)	5 µL
T7 F(10 µM)	0.3 µL
T7 TER(10 µM)	0.3 µL
ddH2O	Up to 10 µL

Procedure

Plasmid extraction

FastPure Plasmid Mini Kit (Vazyme) is used

Material:

• Mixture of 150μl RNaseA and 30 mL Buffer P1
• Buffer P2, Buffer P3, Buffer PW1, Buffer PW2
• ddH2O
• FastPure DNA mini Columns
• Collection Tubes(2mL)

Protocol:

• Transfer the bacterial solution into a centrifuge tube, centrifuge at 10,000 rpm for 1min, and discard the culture medium.
• Add 250μl mixture of Buffer P1 and RNaseA and mix with pipette gun
• Add 250μl Buffer P2 and reverse to mix.
• Add 350 μl Buffer P3 and centrifuge at 12,000 rpm for 10min after reversing and mixing.
• Place the FastPure DNA mini Columns into Collection Tubes (2mL), transfer the supernatant to the adsorption column, centrifuge at 12,000 rpm for 60s, remove the liquid in collection tubes (2mL), and put the adsorption column back to the collection tube.
• Add 600 μl Buffer PW2 (diluted with absolute ethanol), centrifuge at 12,000 rpm for 60s, discard the waste liquid, put the adsorption column back to the collection tube, and repeated once.
• Dry the adsorption column by centrifugation at 12,000 rpm for 1min.
• Put the adsorption column into a new 1.5mL centrifuge tube. Add 100μl ddH2O to the center of the adsorption column. After 2min, centrifuge at 12,000 rpm for 1min.
• Discard the adsorption column and store the products in the centrifuge tube in the -20℃ refrigerator.

Protein expression

Material:

• Recombinant plasmid
• E. Coli BL21 (DE3) competent cells
• LB medium
• IPTG
• Buffer A

Protocol:

• Transform the recombinant plasmid into E. Coli BL21 (DE3) competent cells. Select the monoclonal on the transformation solid medium into LB liquid medium and incubate it overnight at 37℃ and 220rpm as seed liquid.
• Transfer the seed liquid at 1:50 and incubate at 37℃ and 220 rpm. When the bacteria grew to OD600 of 0.6 ~ 0.8, add and express IPTG with a final concentration of 1 mm at 16℃ for 24 hours.

Cell crushing and sample preparation

Material:

• BufferA
• SDS Loading Buffer

Protocol:

• After induction and expression, measure the OD600 of the bacterial liquid, centrifugate it at 8,000rpm for 2min, collect the bacteria, and add Buffer A to resuspend the bacteria. Use a high-pressure crusher to crush the cells.
• Suck 80 μ L sample and centrifugate it at 12,000rpm for 1min, then suck 80μl supernatant as sample. Abandon the other supernatant, resuspend the deposit by 420μl Buffer A and suck 80μl as the bacterial crushed precipitated sample.
• Add 20 μL 5×SDS loading buffer to each sample (2.), mix well, place in boiling water for 10min, and centrifuge at 12,000 rpm for 1min.

SDS-PAGE electrophoresis and staining

Material:

• Distilled water,
• 30% ACR BIS
• Tris
• pH8.8
• 10% SDS
• 10% ammonium persulfate
• TEMED,
• staining solution
• decolorizing solution

Protocol:

• Preparation of separating glue: assemble four clean thick (1.5 mm) and thin glass plates into the glue maker one by one, configure 12% separating glue, inject it between the two glass plates, flatten the liquid surface with isopropanol, and stand for 45min until it solidifies.
• Preparation of concentrated gel: pour out isopropyl alcohol, prepare 5% concentrated gel, inject it above the separation gel, insert the comb, and preserve for 45min until it solidifies.
• Loading: take out the gel and install it in the vertical electrophoresis tank. Dilute 5×Tris Glycine to the working concentration and pour it into the internal and external electrophoresis tanks. Carefully remove the comb and spot the sample into the sample hole in sequence. The loading amount is 10 μl.
• Gel electrophoresis: after the electrophoresis equipment is installed, conduct electrophoresis at 80V for 20min. When the bromophenol blue indicator band of the sample migrates to the separation gel, electrophoresis is performed at 120V for 80min.
• Dyeing: take out the gel, put it into the container, add the dyeing solution, and place it in the bleaching shaker for dyeing for 30min.
• Decolorization: discard the staining solution, add the decolorizing solution, put it in the decolorizing shaker, and change the decolorizing solution every 30min until the protein staining band is clear and the background color disappears.

Protein purification and concentration

Material:

• Ni-NTA 6FF（5mL）pre-assembled gravity column
• Buffer A
• Buffer B
• ethanol
• Amicon® Ultra-15 centrifugal filter (Millipore)

Protocol:

Use Ni-NTA 6ff (5mL) pre-assembled gravity column on supernatant sample, purify the protein by nickel affinity chromatography. During purification, the flow rate of the chromatographic column sample should be less than 5mL/min. The specific purification steps are as follows:

• Discharge the liquid in the chromatography column and wash the chromatography column with ddH2O for 5-8 column volumes.
• Balance 5-8 column volumes with Buffer A.
• Inject the protein supernatant sample into the chromatography column, and collect the effluent of the chromatography column during the process, and the sample is the permeate.
• Use Buffer A to wash the chromatographic column for 5-8 column volumes and collects the discharged liquid, which is the equilibrium liquid.
• Use different concentrations of Buffer B (10%, 20%, 30%, 50%, 100%) for gradient elution, and collect the effluent.
• Wash the chromatographic column with ddH2O after elution, and wash 5-8 column volumes.
• Wash 5-8 column volumes with 20% ethanol and store them in 20% ethanol at 4℃.
• Apply the samples to SDS-PAGE gel electrophoresis in order to check the purification effect.
• Place the purified protein in Amicon® Ultra-15 centrifugal filtration unit (Millipore), centrifuge it at 4℃ and 4,500×g, discard the filtrate in the collection pipe, and add PBS buffer to the upper layer of the device. Repeat several times until the effect of changing protein buffer is achieved.
• Gently mix and take out the protein concentrate, and add glycerol to the final concentration of 15%. Measure the protein concentration with BCA kit. Refer to the instructions for specific steps. Use SDS-PAGE electrophoresis to check the concentration effect, and 0.22μM filter membrane for sterilization, subpackage and store at - 80℃.

Material:

• Escherichia coli K12 MG1655
• Vibrio parahaemolyticus ATCC 17802
• Staphylococcus aureus ATCC 43300
• enterica serovar Pullorum str. ATCC 9120
• LB medium.
• Fusion proteins purified from previous experiments.

Concentrations of fusion proteins purified from previous experiment

Protocol:

• 1.Take out Escherichia coli K12 MG1655, Vibrio parahaemolyticus ATCC 17802, Staphylococcus aureus ATCC 43300, Salmonella enterica subsp. enterica serovar Pullorum str. ATCC 9120 bacterial seed from the -80℃ refrigerator and put them on ice for melting. In the biosafety cabinet scribe the bacterial liquid on LB solid medium and culture it at 37℃ for 16-24h.
• Select the single colony on the plate, inoculate it into LB liquid medium, and culture it at 37℃ for 10h.
• Collect 200μl bacteria cultured overnight for 10h, centrifuge at 8,000rpm for 2min, discard the medium, wash twice with PBS, and add 200μl LB to resuspend the bacteria.
• Fully mix 100μl fusion protein and 200μl of resuspended bacteria solution, centrifuge it at 37℃ for 30min at 8,000rpm for 2min.
• Remove the supernatant, wash with PBS and collect the precipitate.
• Add 200μl PBS to resuspend the cell pellet and measure the fluorescence value of the cell with a microplate reader.

Material:

• Carboxyl magnetic beads 1mL(50mg/mL)
• Activation buffer 15mL
• Coupling buffer 15mL
• EDC 25mg
• Sulfo-NHS 25mg
• Sealing liquid 15mL
• Storage solution 15mL

Protocol:

• Preparation

  Take an appropriate amount of magnetic beads into a clean EP tube, and mix the magnetic beads again with a vortex meter for 15s.

  Place the EP tube on the magnetic rack for 30s and suck the supernatant with a pipette.

  Remove the EP tube from the magnetic rack, add 1mL of activation buffer and mix.

  Repeat step 2.

  Repeat step 3. and 4. twice.

• Activation

  Prepare 50mg/mL EDC and 50mg/mL sulfo NHS with activation buffer.

  Add 1.5mL of activation buffer, 0.5mL of EDC and 0.5mL of sulfo NHS into the EP tube containing magnetic beads, mix well and store at room temperature for 15min.

  Place the EP tube on the magnetic rack for 30s and suck the supernatant with a pipette.

• Coupling

  Transfer an appropriate amount of protein into the tube and mix with magnetic beads (50μg RBP:100μl magnetic beads), add 2mL of coupling buffer and mix well.

  Put it on the rotator and store it at room temperature for 90min.

  Place the EP tube on the magnetic force for 30s, and suck the supernatant with a pipette.

• Enclosure

  Add 5mL of blocking solution into the EP tube, mix well, put it on the rotator and store it at room temperature for 30min.

  Place the EP tube on the magnetic force for 30s, and suck the supernatant with a pipette.

  Add 5mL of blocking solution into the EP tube, mix well, put it on the magnetic rack for 30s, and use a pipette to suck the supernatant.

  Add 5mL of preservation solution into the EP tube, mix well, put it on the magnetic rack for 30s, and use a pipette to suck the supernatant.

  Repeat 3. and 4. twice.

  Add 1mL of preservation solution into the EP tube and store it in a 4℃ refrigerator.

Material:

• Pathogenic bacteria after activation (109CFU/mL): EC, VP, SA, and SE bacteria
• LB broth medium
• PBS
• magnetic globin complex

Protocol:

• Mix 10μl of pathogenic bacteria and 990μl of PBS.
• (Control group) Shake the solution obtained in step 1 and repeat the step 1 until the bacterial solution is diluted to 103CFU/mL.
• (Experimental group - single bacteria) Shake the solution obtained in step 1, mix 10μl of it with 100μl of magnetic globin complex evenly and add 890μl LB broth medium.
• (Experimental group - mixed bacteria) Shake the solutions of the four bacteria strains obtained in step 1, suck 10μl of each solution, mix them with 100μl of magnetic globin complex evenly and add 860μl LB broth medium.
• Put the centrifuge tubes obtained in step 3 and 4 into a 37℃ 220rpm shaker and shake it for half an hour.
• (Control group) Shake the final solution of step 2 and suck 100μl of it, smear it on LB medium, and put it into a 37℃ incubator for one day and one night.
• (Experimental groups) After the end of the step 5, adsorb the magnetic beads to one side with a magnet, suck away the liquid in the centrifuge tube, then add 1,000μl of PBS, use shaker to resuspend.
• (Experimental groups) After resuspension, suck 10μl of it and 990μl of PBS to mix, shake and mix, suck 100μl of it and paste it on medium, and put it into 37℃ incubator for one day and night.
• (Control group) Record the number of single colonies on the obtained medium at step 6 “n” and the number of single colonies on the obtained medium at step 8, “M”. Calculate the separation rate by the equation: M/n × 100%.

Material:

• Whatman 1442-047(filter paper)
• 5%BSA
• Polymyxin B(10mg/mL)
• ddH2O

Chromogenic substrates

Name	Bacteria	Enzyme	Color variation	Concentration
CPRG	EC	β-gal	yellow→red	25mg/mL
X-β-glu	VP	β-glu	colorless→blue	100mg/mL
pNPG	SA	α-glu	colorless→yellow	100mg/mL
magenta caprylate	SE	esterase	colorless→violet	100mg/mL

Protocol:

• Steep Whatman 1442-047 in 5%BSA at 4℃.
• After one night, discard BSA and wash the filter paper with ddH2O until no foam.
• Dry the filter paper at 55℃.
• Take out the dried filter paper and use a punching machine to punch out roundish paper(diameter=6mm).
• Use tweezers to place roundish paper into a 96-well plate, add 1μl chromogenic substrate, 1μl Polymyxin B (10mg/mL) and 5μl ddH2O.
• Preserve the pretreated paper at 37℃ for 0.5-1h.

Material:

• Polymyxin B
• Chromogenic substrate
• PBS
• Bacteria

Protocol:

• Add 0.5μl chromogenic substrate, 0.2μl Polymyxin B (10mg/mL) and 20μl bacteria (109CFU/mL, resuspended by PBS) to a 0.2mL centrifugal tube.
• Preserve at 37℃ for 2h,then take out the tube to check the color.

Material:

• Paper-base sensor
• PBS
• Bacteria

Protocol:

• Dilute solution of bacteria (10⁹ CFU/mL) to 10⁸,5×10⁷,10⁷,5×10⁶,10⁶...... 10¹ CFU/mL.
• Add 10μl of diluted solution to a paper-based sensor, then preserve at 37℃ for 2h.