Experiments/Protocols

1  Preparation of the experiment

1.1  Preparation of LB liquid  culture medium

(1)  Mix 1% NaCl, 1% peptone, 0.5% yeast extraction, and ddH₂O into in glass bottle or flask.

(2)  Mix well and autoclave on liquid cycle at 121°C for 20  min to sterilize.

(3)  Allow medium to cool after autoclaving.

(4)  Store the LB liquid  culture medium at room temperature or 4°C.

1.2  Preparation of LB solid culture medium

(1)  Mix 1% NaCl, 1% peptone, 0.5% yeast extraction, 1.5%   agar , and ddH₂O into in glass bottle or flask.

(2)  Mix well and autoclave on liquid cycle at 121 °C for 20  min to sterilize.

(3)  Allow medium to cool to 50-60 °C after autoclaving.

(4)  Aseptically pour plates in a laminar flow hood. Allow to solidify.

(5)  Store prepared LB agar plates at 4 °C.

1.3  Strain inoculation

(1)  Inoculate the strains synthesized by the company, which were transformed into the plasmids (containing target fragments) into 5 mL of liquid LB culture medium, respectively.

(2)  Overnight incubation at 37°C, 220 rpm.

 

2  Plasmid construction and transformation

2.1  Preparation of LB liquid  culture medium Plasmid extraction

In this section, we used the DiaSpin Plasmid Mini-Preps Kit to extract four plasmids:

A.  pUC57-DarL-BsjL-His-DarA-BsjA,

B.  pUC57-DarL-NisL-His-DarA-NisA-DarE,

C.  pUC57-DarE-BsjM and

D.  pUC57-NisB-NisC-DarE

The procedure is as follows ：

(1)  Equilibrate column: Add 500 μL Buffer S to balance the Miniprep column, and centrifuge at 12,000 ×g for 1 min.

(2)  Collect 5 mL of overnight overnight LB bacteria culture. Centrifuge at 8,000 ×g for 2 min to pellet the bacteria and discard the supernatant.

(3)  Add 250 μL Buffer SP1 (containing RNaseA) to suspend the bacteria completely by vortexing.

(4)  Add 250 μL Buffer SP2, and gently invert the tube 5-10 times.

(5)  Add 350 μL Buffer SP3 and immediately gently invert the tube 5-10 times. Centrifuge at 12,000 ×g for 5-10 min.

(6)  Transfer the supernatant to the column, centrifuge at 8,000 ×g for 30 sec, and discard the filtrate in the collection tube.

(7)  Add 500 μL Wash Solution, centrifuge at 9,000 ×g for 30 sec, and repeat this step.

(8)  Place the column in a clean 1.5 mL tube. Add 50 μL Elution buffer to the center of the membrane. Incubate for 1 min at room temperature. Centrifuge at 9,000 ×g for 1 min and keep the DNA solution.

 

2.2  Amplification of target fragments by PCR

The reaction system and procedure for PCR amplification of fragments are shown in the table below:

 

The PCR amplified fragment information is listed below:

 

2.3  Agarose gel electrophoresis

(1)  Preparation of 1× TAE solution

Dilute 50× TAE concentrates to a working concentration (1×), i.e., add 20 mL of the 50× TAE concentrates mix to 980 mL of ddH₂O.

(2)  Preparation of 1% DNA agarose gel

a)  Weigh 0.6 g agarose powder using a balance.

b)  Pour 60 mL 1× TAE solution using a measuring cylinder.

c)  Add the 1× TAE solution and agarose powder into a conical flask and mix them well.

d)  Cover the conical flask and heat it in a microwave until all powder is dissolved completely.

e)  Cool agarose solution to about 60 °C and add YeaRed™ nucleic acid gel stain to a final concentration of 0.1 μL/mL.

f)  Pour gel solution into gel tray and insert well comb. Wait for 20-30 min until the gel is solidified.

(3)  Electrophoresis conditions

a)  Remove the comb and place the solidified agarose gel into the horizontal electrophoresis Geltank.

b)  Pour the TAE solution into the Geltank, and make sure the solution covers the surface of the agarose gel.

c)  Prepare DNA samples by mixing the 10× DNA loading buffer with PCR products at a ratio of 1: 9.

d)  Load 5 μL DNA marker and 3.5 μL PCR products into wells of solidified agarose gel.

e)  Run gel electrophoresis at 180 V in 1× TAE buffer until sufficient migration (at about 25-30 min).

f)  Visualize DNA bands under UV light.

 

2.4  Fragment extraction

(1)  Cut the DNA gel:

a)  Excise DNA bands of interest quickly using a clean scalpel under UV light.

b)  Cut a minimal amount of agarose gel to minimize contaminants.

c)  Transfer gel slices into pre-weighed microcentrifuge tubes and determine the weight of gel slices.

(2)  DNA gel Extraction: 

In this section, we used the DiaSpin DNA Gel Extraction Kit to extract the DNA fragments, the procedure is as follows ：

a)  Add 3 times of sample volumn of Buffer B2, and heat at 50 ℃ for 10 min until the gel is completely dissolved.

b)  Transfer the solution into columns, centrifuge the tubes at 8000 ×g  for 1 min, and discard the filtrate in the collection tube.

c)  Add 500 µL Wash Solution centrifuge the tubes at 9000 ×g  for 1 min, and discard the filtrate in the collection tube.

d)  Repeat the wash step.

e)  Place the column in a clean 1.5 mL tube. Add 30 μL Elution buffer to the center of the membrane. Incubate for 1 min at room temperature. Centrifuge at 9,000 ×g  for 1 min and keep the DNA solution.

 

2.5  Double enzyme digestion of fragments and vectors

The digestive sites of the fragments and vectors are shown in the table below:

 

The reaction system and procedure for double enzyme digestion are shown in the table below:

 

2.6  Agarose gel electrophoresis of digest product of vectors

This procedure is described previously.

2.7  Ligation of recombinant plasmids

The reaction system and procedure for ligation of vector and fragment are shown in the table below:

 

After the enzyme ligation reaction, we will get the following four recombinant plasmids:

Plasmid A ： pRSFduet-DarL-BsjL-His-DarA-BsjA

Plasmid B ： pRSFduet-DarL-NisL-His-DarA-NisA-DarE

Plasmid C ： pETduet-DarE-BsjM

Plasmid D ： pETduet-NisB-NisC-DarE

2.8  Transformation of recombinant plasmids

In this step, we transformed the plasmids A and B into BL21(DE3) competent cells, respectively. The procedure is as follows:

(1)  Thaw BL21(DE3) competent cells on ice.

(2)  Add 5 μL  recombinant plasmids to the 50 μL  BL21(DE3) competent cells and gently mix. Do not vortex.

(3)  Incubate the mixture of plasmids and competent cells on ice for 30 min.

(4)  Heat shock the cells at 42 °C for 90 sec.

(5)  Immediately return the mixture to the ice for 2 min.

(6)  Add 950 μL  LB culture medium without antibiotics into the mixture.

(7)  Centrifuge the tubes at 4000 ×g  for 2 min to pellet the cells.

(8)  Remove 600 μL of supernatant and resuspend the cells in the remaining LB culture medium.

(9)  Spread 150 μL cells onto LB agar plates containing appropriate antibiotics.

(10)  Incubate the solution at 220 rpm, 37 ℃ for 60 min.

(11)  Culture in a petri dish overnight at 37 ℃.

 

2.9  PCR identification of transformants

(1)  Pick 8 well-isolated colonies from the transformation plate onto a new LB agar plate, and incubate at 37 ℃ for 6 h.

(2)  Pick the colonies, resuspend them in 20 μL of sterile water, and the resuspension is lysed at 98 °C for 10 min.

(3)  Centrifuge the tubes for 5 min to pellet the cell debris.

(4)  Add 1 μL of the supernatant as a DNA template and followed by PCR amplification according to the following table :

(5)  After amplification, analyze 3.5 μL of the PCR reaction by agarose gel electrophoresis. The procedure is as previously described.

(6)  If the target band appears, it indicates that the plasmid has been successfully transformed into the BL21(DE3) competent cells.

(7)  Inoculate the positive transformants in an LB culture medium containing antibiotics at 220 rpm, 37 ℃ overnight.

 

2.10  Preparation of Competent Cells

In this step, we need to prepare competent cells containing A (or B) plasmid, and then transform the C (or D) plasmid into them. The procedure is as follows:

(1)  Inoculate overnight cultures 1: 100 into 50 mL fresh LB culture medium in conical flasks. Incubate at 37°C, 220 rpm until OD₆₀₀ reaches 0.3-0.5.

(2)  Transfer the culture solution into the centrifuge tube and place it on the ice for 20 min.

(3)  Centrifuge at 5,000 rpm at 4 ℃ for 5 min and discard the supernatant.

(4)  Resuspend the pellet gently in 10 mL of pre-cooling 0.1 M CaCl₂ and ice bath for 30 min. Centrifuge as before and discard the supernatant.

(5)  Resuspend the pellet gently in 2 mL of pre-cooling 0.1 M CaCl₂ containing 10% glycerol.

(6)  Aliquot competent cells into pre-chilled tubes, 100 μL per tube.

(7)  Flash freeze in dry ice and transfer to -80°C for long-term storage.

 

2.11  Transformation of another plasmids and PCR identification

In this step, we transform the C (or D) plasmid into previously prepared competent cells containing A (or B) plasmid, respectively. The transformation steps described previously.

PCR identification of the transformants required the use of two pairs of primers specifically paired with different plasmids for fragment amplification, and only transformants with two observed bands contain both plasmids successfully. The PCR reaction system and procedure are described previously.

 

3  Protein expression and purification

3.1  Protein expression

(1)  Inoculate the positive transformants in an LB culture medium containing antibiotics at 220 rpm, 37 ℃ overnight.

(2)  Inoculate overnight cultures 1: 100 into 200 mL fresh LB culture medium containing antibiotics in conical flasks.

(3)  Incubate at 37°C, 220 rpm until OD₆₀₀ reaches 0.8.

(4)  After the temperature of the LB bacterial medium is reduced to 18 ℃, add IPTG to a final concentration of 0.8 mM.

(5)  Induction at 18 ℃, 200 rpm for 20 hours.

(6)  Harvest cells by centrifugation at 4,000 rpm for 10 min at 4 °C. Discard supernatant.

 

3.2  Protein purification by affinity chromatography on Ni-NTA resin

(1)  Resuspend the pellet in lysis buffer by adding 10 mL of lysis buffer (containing 8 M urea) to 1 g of wet-weight bacteria.

(2)  Sonication of the bacterial resuspension is performed using φ6 probes at 30% power for 10 min with 3 sec on and 3 sec off.

(3)  Centrifuge at 12,000 rpm at 4 ℃ for 20 min to remove debris.

(4)  Resuspend 50% His-tag purification resin gently, take 1 mL resin, and centrifuge at 1,000 rpm, 4 °C for 30 sec to discard the storage solution.

(5)  Equilibrate the resin by adding 1-5 bed volumes of denaturing lysis buffer and mixing well, centrifuge again, and discard the supernatant.

(6)  Mix the resin with lysate supernatant and incubate for 60 min with gentle agitation on a side or horizontal shaker at 4 ℃ .

(7)  Load the mixture of lysate and resin into an empty chromatography column.

(8)  Remove the snap-off cap at the bottom of the column, and allow the liquid to flow out of the column by gravity.

(9)  Wash the resin with 1-2 bed volumes of denaturing lysis buffer (containing 8 M urea) and allow the liquid to flow out by gravity. Repeat washing 5 times.

(10)  Elute protein 5-10 times with denaturing elution buffer (containing 8 M urea), with one-bed volume of denaturing elution buffer each time.

(11)  All flow-out components need to be identified by SDS-PAGE analysis.

 

3.3  Preparation of SDS-PAGE gel

In this section, we used the SDS-PAGE Color Preparation Kit to prepare the SDS-PAGE gel. Take the example of preparing a piece of gel, and the procedure is as follows :

(1)  Assemble the gel casting stand.

(2)  Prepare to resolve gel by adding 2.7 mL of 2× gel solution and 2.7 mL of 2× resolving gel buffer, and mix evenly.

(3)  Add 55 μL modified polymerization accelerator to the mixture. Mix by gently swirling to avoid bubbles.

(4)  Pour the resolving gel solution into the casting stand 1.5 cm from the top. Layer with water to ensure the  surface is without bubbles.

(5)  Allow to polymerize at 25 °C for 10 min until distinct interface forms.

(6)  Prepare stacking gel by adding 0.75 mL of 2× stacking gel solution and 0.75 mL of 2× colored stacking buffer, and mix evenly.

(7)  Add 15 μL modified polymerization accelerator. Mix by gently swirling to avoid bubbles.

(8)  Pour stacking gel on top of resolving gel to the top of glass plates.

(9)  Insert comb slowly to avoid bubbles.

(10)  Allow to polymerize for 10-15 min. Carefully remove comb for the further SDS-PAGE analysis.

 

3.4  SDS-PAGE analysis of protein

(1)  Install the SDS-PAGE gel into the vertical electrophoresis holder.

(2)  Prepare protein samples by mixing with 5× SDS sample loading buffer at a ratio of 1: 4, and boiling at 98 ℃ for 5 min.

(3)  Load 6 μL of protein sample per well and 4 μL of protein ladder in a separate well.

(4)  Assemble the SDS-PAGE apparatus and fill it with 1× Tris-glycine SDS running Buffer.

(5)  Run at 90 V through stacking gel then increase to 120 V.

(6)  Stop when the dye front reaches the bottom of the gel.

(7)  Carefully remove gel and stain with Coomassie blue R-250 for 60 min with gentle agitation.

(8)  Destain gel with destain solution (containing 40% ethanol and 10% acetic acid) until protein bands are visible and the background is clear.

 

4  Structure and activity verifications of proteins

4.1  Detecting the structures of purified protein

(1)  Filter the collected eluate through a 0.45 μm filter membrane.

(2)  Purify by RP-HPLC using a C4 column, with mobile phase A: water with 0.1% trifluoroacetic acid (TFA) and mobile phase B: 80% acetonitrile with 0.1% TFA.

(3)  Lyophilize the precursor peptides then dissolve them in deionized water.

(4)  Filter through a 0.22 μm filter membrane.

(5)  Detect the molecular weight of each precursor peptide using Q-TOF/MS.

(6)  Transfer 30 μL of the peptide solution into a glass vial lined with a plastic insert, avoiding the formation of bubbles.

(7)  Select the ESI ion source and positive ion mode on the mass spectrometer.

(8)  Inject a 5 μL sample volume, with mobile phase A: water with 0.1% TFA and mobile phase B: 80% acetonitrile with 0.1% TFA.

 

4.2  In vitro cleavage on proteins

In this step, the purified protein needs to be cleaved by protease (lysyl endopeptidase) and the released core peptide is the final mature product. The procedure is as follows ：

(1)  Equilibrate the powder of lysyl endopeptidase in a drying oven for at least 30 min.

(2)  Dissolve powder of lysyl endopeptidase in a buffer (20 mM Tris-HCl, pH 8.5) to a concentration of 200 AU/mL.

(3)  Add lysyl endopeptidase to the purified protein in a 100 AU: 1 g ratio.

(4)  Incubate at 37 °C for 4 h.

 

4.3  Agar diffusion growth inhibition assay

(1)  The indicator strain B. subtilis 168 is cultured overnight at 37 °C in a liquid LB culture medium.

(2)  Agar plates are prepared by mixing 20 mL melted agar (ca. 42 °C) with the cell cultures (1%, v/v) and poured into a sterile petri dish immediately.

(3)  All core peptides are dissolved in sterile ddH₂O to a stock concentration of 100 μM.

(4)  10 μL of the stock solutions are separately spotted for the agar diffusion growth inhibition assay. The sterile ddH₂O is used as the negative control.

(5)  Plates are incubated at 37 °C for 12 h after being spotted.

(6)  The antimicrobial activity is determined by the presence/size of the growth inhibition zones.