Part:BBa_K3838789

J-FabV

Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Literature has improved that Vibrio cholera FabV, a functional homologue of E.coli FabI, can be used as a suitable marker for the selection and maintenance of plasmid vectors in E.coli. Triclosan is FDA-approved as a safe, broad-spectrum antibacterial. It is widely used in household chemicals such as soap and toothpaste and has also been found in human breast milk and urine. By expressing resistance genes, we gave triclosan resistance to engineering bacteria and achieved screening effect.

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
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Data:SZU-China 2021 TEAM

1. The DNA level

After the engineered bacteria were transformed, the target plasmid after DH5α transformation was extracted and verified by DNA gel electrophoresis. For Nissle 1917, we extracted the target plasmid and amplified the SOD expression element on the plasmid, which was verified by DNA gel electrophoresis. We also carried out enzyme digestion verification on the plasmid, adding restriction endonuclease MIuI and SacI for double enzyme digestion, and obtained bands of the expected size, as shown in figure 1. This further indicates that our plasmid transformation is successful.

Figure 1 A. Gel electrophoresis of DNA transformed into EScherichia coli DH5α by plasmid PJSG (5939 bp). B.After transformation, Nissle 1917 SOD expression element was amplified(target fragment size was 1890 bp) C. The PJSG was double-digested with restriction endonuclease MIuI and SacI, and the expected band sizes were 1918 bp and 4021 bp

2. Protein level

The experimental group was DH5α with PJSG plasmid, and the control group was DH5α empty vector. As can be seen from figure 2, protein expression in the experimental group was near 45 kDa, which was consistent with our target protein band of 44kDa. It indicated that FabV protein was successfully expressed by the engineered bacteria.

Figure 2.SDS-PAGE of DH5α intracellular protein containing PJSG plasmid was performed. The untransformed DH5α was used as blank control group. The target protein size was 44 kDa

3. Functional characterization

0.625μM/mL triclosan concentration: there was no colony growth in the control group, but a few colonies grew in the experimental group. We selected a single colony and transferred it to the liquid before plasmid extraction for gel running and enzyme digestion verification. The target band was 5477 bp, proving that the growing colony was indeed a transpromoter. DH5a: In order to find the effective working concentration of this screening system, LB plates were prepared at 0.625μM/mL triclosan concentration 1.25μM/mL triclosan concentration and without resistance, The DH5α bacterial solution at the early logarithmic growth stage was coated on the plate on the medium (the concentration of the two groups was controlled at about OD=0.4 ). Place the plates at 37℃ in a constant temperature incubator for 18h, and obtain the results, as shown in figure 3. Non-resistant group: both kinds of bacteria are overgrown on the plate; 1.25μM/mL triclosan concentration: There was no colony growth in the control group, but a few colonies grew in the experimental group. We selected a single colony and transferred it to the liquid, then plasmid was extracted for gel running and enzyme digestion verification, and the target band was 5477 bp, proving that the growing colony was indeed a transpromoter. To further confirm, triclosan plates with 0.625μM and 1.25μM concentrations were re-selected for plasmid raising validation after the 4C plate was retained for 14 days, and the target bands were also obtained, as shown in figure 4. In conclusion,0.625μM/mL and 1.25μM/mL triclosan concentrations can be used as screening conditions for non-antibiotic DH5α engineering bacteria.

Figure 3 A. Untransformed DH5α coated in LB without triclosan B. Transformed DH5α coated in LB without triclosan C. Untransformed DH5α was coated on LB D. with 0.625μM/ mL triclosan. The transformed DH5α was coated on LB E. with 0.625μM/ mL triclosan. Untransformed DH5α was coated on LB F. The transformed DH5α was coated on LB supplemented with 1.25μM/ mL triclosan

Figure 4 A.the single colony transfer plasmid plasmid obtained by 0.625μM/mL triclosan plate B.the single colony transfer plasmid obtained by 1.25μM/mL triclosan plate. The target plasmid size was 5477bp.

Nissle 1917: Because there are two kinds of chassis in the project, we also conducted a screening concentration determination experiment in Nissle 1917. We adjusted the concentration of triclosan to 2.5 mmol/ml-10 mmol/ml and diluted the coating board 5 times. Figure 5 lists the growth conditions of untransformed Nissle 1917 on plates with different concentrations and the growth conditions of transformed Nissle 1917 on plates with different concentrations.

As can be seen from the figure, the growth conditions of Nissle 1917 (NJB) transformed PJB plasmid transformed by different gradients were better than those of the untransformed control group, indicating that FabV gene had certain stress resistance to triclosan. It provides support for the use of the system as a screening system for non-antibiotics. At the concentration of 2.5 mmol/mL,the experimental group had obvious bacterial growth and single colony, while the control group almost did not grow on the plate. Therefore, a plate with a concentration of 2.5 mmol/mL was selected for PCR after single colony transfer liquid and plasmid extraction, and the target band size was 1756 bp, as shown in figure 6. The sample band was consistent with the target band. It was proved that the bacteria growing on the experimental plates were NJB engineered bacteria, which proved that the non-antibiotic screening system of triclosan /FabV was also suitable for Nissle 1917.

Figure. 5 A. The untransformed Nissle 1917 coated on LB without triclosan. B. The transformed Nissle 1917 was coated on LB without triclosan.C. The untransformed Nissle 1917 was coated on LB with 2.5 mmol/mL triclosan.D. The transformed Nissle 1917 was coated on LB with 2.5 mmol/mL triclosan.E. The untransformed Nissle 1917 was coated on LB with 5 mmol/mL triclosan.F. The transformed Nissle 1917 was coated on LB with 5 mmol/mL triclosan. G. The untransformed Nissle 1917 was coated on LB with 10 mmol/mL triclosan.H. The transformed Nissle 1917 was coated on LB with 10 mmol/mL triclosan.

Figure 6. The target band size of PCR amplification was 1756 bp, and the actual band size was consistent with the target band.