Part:BBa_K5101002

Plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7

Nitric-oxide-inducible antimicrobial peptide expression plasmid

Usage and Biology

This composite plasmid, pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7, is engineered for precise expression control in response to oxidative stress conditions. It incorporates a dual-promoter system where the first part consists of a strong constitutive promoter J23119 for consistent expression of eGFP, serving as a reporting system. Following the pJ23119, a nitric-oxide-inducible promoter system, SoxR/SoxS, is used to control the expression of an antimicrobial peptide (AMP), enabling specific activation under stress conditions. The plasmid is equipped with Ribosome Binding Sites (RBS) for optimal translation efficiency of both proteins, ensuring robust expression in E. coli Nissle 1917. The T7 terminator ensures transcriptional termination, maintaining system stability and efficiency.

Construction of the plasmid

We designed the plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 as shown in Figure 2-1, and using homologous recombination integration, we constructed the expression plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-AMP-T7. We transformed it into DH5α and extracted the recombinant plasmid after picking several single colonies of E. coli on the transformed plates for inoculation. We performed PCR to verify that the primers were specific primers with a target fragment of 2019bp, and the results are shown in Figures 2-2. We sent the plasmids with correct band positions to GENEWIZ Co. for sequencing. As shown in the figure 2-3, the sequencing results were all correct, which verified that the recombinant plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 was successfully constructed.

Figure 2-1 NO-inducible plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7

Figure 2-2 M：DL2000 DNA Marker(Vazyme)

plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7(2019bp)

Figure 2-3 plasmid pET29(a)-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7 sequencing result

Protein expression validation

Figure 3-1-1 Process of AMP protein purification by nickel columns

Figure 3-1-2 Process of performing SDS-PAGE to verify the AMP protein

SDS-PAGE validation

We extracted the correctly sequenced NO-inducible plasmid and transformed it into EcN for expression. Since NO is hazardous, we utilized SNP (sodium nitroprusside) as a nitric oxide (NO) donor for inducible expression. SNP can decompose and release NO in the presence of light, thus initiating downstream gene expression.

We picked a single colony, added 5ml LB culture solution in a test tube, added one thousandth (5µl) of the resistance gene Kana, picked the single colony into the test tube, and put it into a 37℃ incubator at 220rpm to incubate for 16 hours. Two tubes of bacteria were raised as experimental group and control group; OD₆₀₀ of the bacterial solution was measured, and the OD₆₀₀ of the bacterial solution was diluted to 0.1 (0.1×(50+colonization amount) = OD₆₀₀×colonization amount), which was added into 250ml conical flasks with 50ml of LB+50µl of Kana, and the culture was amplified for four to five hours, until both flasks of the bacterial solution had an OD₆₀₀ value close to 1.0. We initially set the induction temperature at 16°C and the induction time at 24 hours, and the final concentration of SNP (sodium nitroprusside) 100µM was replenished every 12 hours. At the end of induction, the protein fragmentation step was carried out, 200 µl of total protein samples from experimental and control groups were dispensed before ultrasonic fragmentation; after fragmentation, 200 µl of supernatant was aspirated as post-fragmentation supernatant, and 200 µl of precipitate was resuspended in ultrapure water as post-fragmentation precipitate. Concentrate by ultrafiltration, take up 200µl of concentrated supernatant as a sample; protein gel sample, 5-10µl for each well site, electrophoresis running gel; Thomas Brilliant Blue medium-high fire staining for two minutes, decolorization.

Because the Marker indication range is 8-200KD, and the AMP size is only 8.5KD after adding His tag, so the bands are not obvious. Therefore, we performed Western Blot protein blotting (WB) to verify whether the protein was expressed.

Figure 3-1-3 SDS-PAGE characterization of antimicrobial peptide containing NO-inducible promoter

M: Protein Marker; 1: Whole bacteria after induction; 2: Supernatant after induction and sonication; 3: Precipitate after induction and sonication

Western Blot Initial Validation

According to the previous results, we ran the remaining protein samples on the gel, transferred them, and conducted the WB experiment. We applied the primary antibody (1:7000) overnight, washed with TBST, applied the secondary antibody (1:5000) for 1 hour, continued washing, prepared the protein-developing solution in a 1:1 ratio, and developed the results. The results are shown in Figure 3-2-1. We found that the baseline expression level of AMP was relatively high, and inclusion bodies were formed with a high content in the precipitate. We decided to characterize the NO-inducible promotor SoxR/SoxS to determine the optimal inducer concentration and induction time, optimize the conditions, and obtain more AMP than the baseline expression.

Figure 3-2-1 Western Blot result of AMP expression of plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-eGFP-RBS-AMP-T7

1-3 represent uninduced total protein, concentrated supernatant, and precipitation, respectively; well M is the Marker (at 8KD); 4-6 represent induced total protein, concentrated supernatant, and precipitation, respectively.

Western Blot Re-validation

Through the characterization of the promoter SoxR/SoxS (more details of the characterization can be found on the main page of BBa_K554000), we got the optimal induction time of p-SoxR-pSoxS-eGFP-RBS-AMP plasmid in EcN is 10 hours, and the optimal induction concentration of SNP is 100µM.

So we carried out the induction of AMP expression according to the above experimental conditions, picked single colonies, added 5 ml of LB culture medium to the test tube, added one thousandth (5µL) of the resistance gene Kana, picked the single colonies into the test tube, and put them into the 37℃ incubator at 220rpm for 16 hours. Two tubes of bacteria were raised as experimental group and control group. Measure the OD₆₀₀ of the bacterial solution, dilute the OD₆₀₀ of the bacterial solution to 0.1 (0.1×(50+colonization amount)=OD₆₀₀×colonization amount), add it into 250ml conical flasks of 50ml LB+50µl Kana, and amplify the incubation for four to five hours until the OD₆₀₀ values of both flasks were close to 1.0. Set the induction time to 10 hours, and SNP (sodium nitroprusside) 100µM final concentration was added. Protein fragmentation step was carried out at the end of induction, 200 µl of total protein samples from experimental and control groups were dispensed before ultrasonic fragmentation; after fragmentation, 200µl of supernatant was aspirated as post-fragmentation supernatant sample solution, and 200µl of precipitate was resuspended in ultrapure water as post-fragmentation precipitate. Take 20µl of sample plus 5µl of SDS-PAGE protein sample buffer (5×) cook protein at 100℃ for 10min, protein gel sample, run the gel; run the gel of the rest of the protein sample, transfer the membrane, and do the WB experiment; apply the primary antibody (1:7000) overnight, wash with TBST, apply the secondary antibody (1:5000) for 1hour, continue to wash, and then 1:1 configure the protein developer solution to develop the image.

As can be seen in Figure 3-3-1 WB development graph, the antimicrobial peptide containing NO-inducible promoter formed inclusion bodies, and the precipitate contained a large amount of AMP, but the induced AMP developed deeper and clearer bands than the background expression level. This indicates that the expression of the antimicrobial peptide mediated by the NO-inducible promoter was successful, and the precipitate contained a large amount of AMP.

Figure 3-3-1 WB characterisation of antimicrobial peptides containing NO-inducible promoter

1-3 are uninduced precipitate, supernatant and total protein, respectively; 4-6 are induced precipitate, supernatant and total protein, respectively; M wells are at Marker 6.5-14.4 KD

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 572
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1708
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1678
  
• 1000
  COMPATIBLE WITH RFC[1000]