Experiments

All plasmids were extracted by the HiPure Plasmid Micro Kit from Magen Co., LTD.

1) Overnight culture was centrifuge at 13,000rpm for 1 min at room temperature. Decant or aspirate and discard the culture media.

2) Resuspend pelleted bacterial cells in 250 μL Buffer P1 and transfer to a microcentrifuge tube.

3) Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 8–10 times.

4) Add 350 μL Buffer P3. Mix immediately and thoroughly by inverting the tube 8–10 times.

5) Centrifuge for 10 mins at 13,000 rpm in a table-top microcentrifuge.

6) Insert a HiPure DNA Mini Column II into a 2.0 mL Collection Tube(provided). Apply 800 μL of the supernatant from step 5 to the column by pipetting. Centrifuge for 30~60 s. Discard the flow through.

7) Recommended: Wash the Column by adding 0.5 mL Buffer PW1 and centrifuging for 30–60 s. Discard the flow through.

8) Wash the column by adding 0.75 mL Buffer PW2 and centrifuging for 30–60 s.

9) Discard the flow through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.

10)Place the Column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50~100 μL Elution Buffer or water to the center of each column, let stand for 1 min, and centrifuge for 1 min.

1) The concentration of plasmid or DNA fragment were confirmed by the Nano Drop 2000C (Thermofisher), then the amount of enzyme will be calculated by the amount of plasmid or DNA fragment added to the digestion;

2) Certain amount of plasmid or DNA fragment were added to the 200 μL microcentrifuge tube;

3) The Restriction enzyme (NEB or Takara) and buffer were added based on the calculation on 1) and mixed;

4) The microcentrifuge tube was incubated in 37 ℃ water bath for 2-3 h.

All DNA fragments (DNA digestion or PCR purification) were pumicated by the HiPure Gel DNA Mini Kit from Magen Co., LTD.

Protocol 1: Pure DNA from agarose gel

1) Excise the DNA fragment from the agarose gel with a clean&sharp scalpel. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5mL microcentrifuge tube.

2) Add 2 volume of Buffer GDP to 1 volume of the gel (100 mg gel approximately 100μL). Incubate at 50~55°C for 10 mins or until the gel has completely melted. Vortex or shake the tube every 2-3 mins during the incubation.

3) Insert a HiPure DNA Mini Column II in a 2mL Collection Tube.

4) Add no more than 750μL DNA/agarose solution from step 2 to the Column. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

5) Repeat Step 4 until all of the sample has been transferred to the column.

6) Add 300μL Buffer GDP to the column and incubate for 1 min. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

7) Add 650μl Buffer DW2 to the column. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

8) Add 650μl Buffer DW2 to the column. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

9) Centrifuge the empty Column at 12,000 g for 2 mins at room temperature to dry the column matrix.

10) Transfer the Column to a clean 1.5mL microcentrifuge tube. Add 25~50 μL Elution Buffer or deionized water directly to the center of the column membrane. Let it sit at room temperature for 2 mins.

11) Centrifuge at 12,000 g for 1 min at room temperature. Store DNA at -20°C.

Protocol 2: Pure DNA from PCR Product or Enzyme Reaction

1) Determine the volume of your sample. Transfer the sample into a clean 1.5 mL microcentrifuge tube.

2) Add 2 volumes Buffer GDP to the sample and mix well.

3) Insert a HiPure DNA Mini Column II in a 2 mL Collection Tube.

4) Add no more than 700 μL of the Mixture from Step 2 to the Column. Centrifuge at 12,000 g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

5) Repeat Step 4 until all of the sample has been transferred to the column.

6) Add 450μL Buffer DW2 to the column. Centrifuge at 12,000g for 1 min at room temperature. Discard the filtrate and reuse collection tube.

7) Repeat Steps 6 for a second Buffer DW2 wash step.

8) Centrifuge the empty Column at 12,000g for 2 mins at room temperature to dry the column matrix.

9) Transfer the Column to a clean 1.5 mL microcentrifuge tube. Add 25-50 μL Elution Buffer or deionized water directly to the center of the column membrane. Let sit at room temperature for 2 mins. Centrifuge at 12,000g for 1 min and store DNA at -20°C.

The PCR For DNA fragment was amplified by 1.1×S4 Fidelity PCR Mix (Genesand) with the following reaction volume: 2 μL for each primers, 2 μL template DNA (10 pg to 10 ng), 44 μL 1.1×S4 Fidelity PCR Mix. For colony PCR, the volume could adjust to 20 μL.

PCR program:

Gibson assembly reaction was conducted by the 2X MultiF Seamless kit (ABclonal)

1) The primers were designed in the snapgene based on the sequence of vector and insert fragment;

2) The insert fragment was prepared by PCR as described in experiment 4;

3) Linearized vector was prepared by digestion as described in experiment 2;

4) The reaction was prepared on the ice with the following volume: linearized vector X μL, insert fragment YμL, 2X MultiF Seamless Assembly Mix 10 μL, ddH2O to 20 μL;

(Note: the amount of linearized vector was recommended to be 0.03 pmol, the ration of insert fragment/ vector is 1:2 (Best recommendation) to 1:3.)

5) Mixed the sample, then incubated at 50℃ for 15mins;

6) Transformation (see experiment 7) and use colony PCR to screen the transformants.

1) Single colony of E.coli strain was selected to LB medium, incubated in the shaker at 37℃, 220 rpm till the OD600 reached 0.3;

2) The cell was transferred to 50 mL centrifuge tube and incubated on ice for 10 mins;

3) Centrifuge 10 mins at 4,000 rpm, 4℃;

3) Linearized vector was prepared by digestion as described in experiment 2;

4) The reaction was prepared on the ice with the following volume: linearized vector X μL, insert fragment YμL, 2X MultiF Seamless Assembly Mix 10 μL, ddH2O to 20 μL;

5) 50 uL/ tube stock at -80 ℃ freezer.

Insert fragment and vector was added at the ration from 3:1-10:1, with at least 0.01 pmol DNA for each, 2 uL T4 ligase buffe, 0.2 uL ligase 4 (i science) and ddH2O added to 20 uL. The sample were incubated at the heating block for 20 mins at 22 ℃.

1) The sample was prepared as follows: 50 μL competent cell prepared as experiment 6, 10 μL ligation sample from experiment 7, 10 μL 5* KCM buffer (0.5M KCl, 0.15M CaCl2, 0.25M MgCl2), ddH2O 20 uL;

2) Incubated on ice for 30 mins;

3) Place at room temperature for 5 mins;

4) Add 1mL LB;

5) Recoved at 37℃ shaker for 45- 60mins, 200-220 rpm;

6) Centrifuge 5 mins at 4,000 rpm, 4℃ and spread the sample with 100 μL sample at suitable plate;

7) Incubate at 37℃ for overnight.

1.4 mL overnight culture and 0.6 mL DMSO were mixed and stocked at -80 ℃ freezer.

Expression of toxic protein and protease inhibitors

The protein was concentrated as the protocol of Millipore ® Filtration Selection Guide.

1) The buffer for SDS-PAGE were prepared as follows:

SDS separating buffer: 18.165 g Tris, 4 mL10%SDS, adjust pH to 8.8 by NaOH, add ddH2O to 100 ml.

SDS stocking buffer: 5.98 g Tris, 4 mL10%SDS, adjust pH to 6.8 by NaOH，add ddH2O to 100 ml.

10 X running buffer (pH8.3): 72 g glycine, 15.15g Tris, 5g SDS, add ddH2O to 500mL, dilute to 1 X running buffer before use.

4 X loading buffer: 0.5 M Tris-HCl (pH6.8) 4.5mL, 10 mL glycerol, 2.3g SDS, 0.004 g bromophenol blue, add ddH2O to 25mL.

Coomassie brilliant blue straining solution: Coomassie brilliant blue 0.25g, methanol 227 mL, acetic acid 46 mL, ddH2O added to 500 mL.

Destraining solution: ethanol 90 mL, acetic acid 20 mL, ddH2O added to 1L.

2) Gel form

3) Gel preparation

Make the separating gel:

Set the casting frames (clamp two glass plates in the casting frames) on the casting stands. Prepare the gel solution (as described above) in a separate small beaker. Wirl the solution gently but thoroughly. Pipet appropriate amount of separating gel solution (about 7-7.5 mL) into the gap between the glass plates. To make the top of the separating gel be horizontal, fill in the gap with 1 mL 75% ethanol. Wait for 20-30min to let it gelate.

Make the stacking gel:

Discard the water and you can see separating gel left. Pipet in stacking gel until an overflow. Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it gelate.

Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow until the buffer surface reaches the required level in the outer chamber.

4) Samples preparation

Mix your samples with loading buffer. Heat them in boiling water for 5-10 mins. Load prepared samples into wells and make sure not to overflow. Don't forget loading protein marker into the first lane. Then cover the top and connect the anodes.

5) Electrophoresis

As for the total running time, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about 1.5 hour for an 80V voltage of a 12% separating gel.

6) Straining and destraining

After electrophoresis, stain with Coomassie brilliant blue staining solution for 2 hours. Discard the staining solution, rinse several times with distilled water, and then add the destaining solution until the protein bands are clear.

Material:

10 X transfer buffer: 144.15g glycine, 30.285g Tris, ddH2O was added to 1 L, and store at 4℃. 1 X transfer buffer: 100 mL 10X transfer buffer, 200 mL methanol, ddH2O was added to 1 L, prepared before use.

10 X TBS buffer (pH7.6): 24.22g Tris, 80g NaCl, adjust pH with 6 mL 12N hydrochloric acid, ddH2O was added to 1 L and store at room temperature.

1 X TTBS buffer (pH7.6): 100 mL 10 X TBS buffer, 1mL Tween-20, dH2O was added to 1 L and store at room temperature.

Blocking solution: 5g skimmed milk powder, 1 X TBST buffer was added to 100 mL and store at 4C.

Experiment Steps:

1) Activate PVDF membrane in methanol for 15 minutes (use a tweezers at all times—do not touch with gloves). Cut membrane and paper almost same size as gel, then soak SDS-PAGE gel (already run) and PVDF membrane and 4 whatman filter papers (cut to size) in Blotting buffer (15 minutes, in fact no time requirement, just wet gel, membrane, filter paper with blot buffer).

2) Assemble sandwich submerged in blot buffer (no bubbles). Black-sponge-two layer of filter paper-SDS/No-SDS PAGE (without stacking gel)- membrane- two layer of filter paper – sponge- white.

3) Quickly transfer sandwich to full assembly filled blot buffer (this buffer should be fresh and cold). Run in the cold room at 90V 350mA for 1 hour or 30V 90mA for overnight.

4) Briefly wash membrane with TTBS to remove methanol.

5) Block the membrane (facing up) with 5% non-fat dry milk in TTBS (could be without Tween 20, since background might high with Tween 20) for 1 hour (or overnight at cold room). Shake well at room temperature for all steps.

6) Incubate with (1:10,000; 1 uL in 10 mL) primary antibody in 5% non-fat dry milk in TTBS for 1 hour.

7) Rinse with TTBS: one quick rinse, 4*10 minutes.

8) Incubate with (1:5,000; 2uL in 10mL) secondary antibody in 5% non-fat dry milk in TTBS.

9) Rinse with TTBS: one quick rinse, 4*10 minutes.

1) The single colony of the engineered bacteria was transferred to 5 mL LB with antibiotic in the working concentration and incubate at the shaker overnight at 37℃ 220 rpm.

2) 2% overnight culture was transferred to 5 mL fresh LB with antibiotic in the working concentration. Arabinose (ara) was added to experimental group with the final concentration of 0.02%. The seed culture was added to control group, but no ara was added. The blank group was set as follows: ara was added at final concentration of 0.02% but no seed culture was added to LB medium. All groups were incubated in a shaker at 37℃, 220 rpm for 6 hours and the OD600 were observed for the results.

1) 200 uL sample and blank control (LB medium) were added to the 96-well plate, and tested by the microplate reader. For eGFP, excitation wavelength was set at 488 nm, while the emission wavelength was 506 nm. For RFP, excitation wavelength was set at 587 nm, while the emission wavelength was 610 nm

2) The OD 600 values of the experimental group, control group, and blank control group were tested in the spectrophotometer, with LB medium as the blank. All experiments were repeat 3 time.

1) The single colony of the engineered bacteria was transferred to 5 mL LB with antibiotic in the working concentration and incubate at the shaker overnight at 37℃ 220 rpm.

2) All overnight culture were measured with a spectrophotometer, and the cell density were adjusted by LB to OD600 = 0.6. All experiments were repeat 3 time.

3) 200 uL samples from the experimental group, control group, and blank control group were added to a sterile 96-well plate, and 3 replicates were set for each group. Control group: wild type TOP10 strain. Experimental group: Engineered bacterial with the corresponding antibiotic at a working concentration and 0.02% ara at the final concentration. Blank group: LB medium without any strain.

4) The 96-well plate were placed into the bioscreen (Log Phase 600, Agilent), with continuous shake at 37℃, 500 rpm. The cell density was tested at OD600, and value was read every hour, and continuous measurement for 20 hours.

5) After the measurement, the results were exported from the computer, and the repeated results of each group were averaged to draw a growth curve.

Strains were grown in LB medium for required time with or without the ara. 20 mL of bacterial supernatant were collected by centrifugation and extracted with an equal volume of ethyl acetate mixed the incubated for 4 h. The organic phase was evaporated, and the residue was dissolved in 1 ml of ethyl acetate and transferred to 1.5 mL microcentrifuge tube. The mixture was dried by Termovap Sample Concentrator and stock at -20℃.

1) The treated sample was dissolved with 200 uL acetonitrile and loaded to HPLC.

2) Using HPLC, the excitation wavelength was set to 210nm, and the measurement time was 20 minutes. The peak time and wave peak of the sample were measured.

1) Different volume of 30 mg/mL Gm stock was added to LB agarose medium to get the LB medium with gradient Gm concentrations of 30/15/10 /5/2/1/0.5/0 μg/mL.

2) The test strains were streaked to the LBA medium with gradient Gm concentrations mentioned above.

3) One group is placed in an anaerobic box (Mitsubishi Gas Chemical Company, INC.), while another group is placed under the aerobic environment. All plates were placed in 37°C incubator for 48 hours.

△dapA/dapA-pUC18T-mini-Tn7T and △dapA were streaked on solid LB medium with or without 200 μM DAP. The plates were tested under aerobic and anaerobic conditions as described above.