Part:BBa_K3771091

PkatG-sfGFP

Description

This composite part consists of promoter P_katG (BBa_K3771047) and sfGFP (BBa_K1321337). The katG promoter (P_katG) is stimulated by oxidative stress, leading to the expression of the downstream gene. sfGFP serves as a reporter to evaluate the strength of P_katG.

Usage and Biology

This composite part was ligated with the pSAA vector and transformed into E. coli. We conducted colony PCR to verify whether E. coli uptake the correct plasmid. The size of the PCR product was as expected. The part has been confirmed by sequencing and has no mutations.

Fig. 1. (A) The electrophoresis result of colony PCR. M: Marker; Lane 1, 2: pSAA-P_katG-sfgfp (2629 bp); Lane 3: pSAA-J23100-sfgfp (2537 bp). (B) Digested the amplified fragments with HindIII. M: Marker; Lane 1: pSAA-J23100-sfgfp; Lane 2, 3: pSAA-P_katG-sfgfp (147, 278, 377, 1933 bp).

We conducted a disk assay to examine what kinds of oxidative stress inducers can P_katG react to. First, 0.5ml of overnight culture was mixed with 3ml of melted 0.5% soft agar LB at 50˚C and overlaid on a LB plate. Next, we put paper discs on the plate and dropped solutions to induce oxidative stress on paper discs. Last, we incubated the plate at 37˚C for 27 hours and checked the result. In our result, P_katG only responds to high-concentration hydrogen peroxide (Fig. 2).

Fig. 2. The disk assay results of P_katG responding to different oxidative stress inducers. H₂O₂s - 30% hydrogen peroxide, 10ul; H₂O₂w - 3% hydrogen peroxide, 10ul; MD - 10 mM menadione, 10ul; DMSO - solvent for menadione, 10ul; PQ - 1 mM paraquat, 10ul; MQ - solvent for paraquat, 10ul.

The promoter strength of P_katG is determined by the expression level of the reporter, sfGFP, under oxidative stress. While using hydrogen peroxide (H₂O₂) as the inducer, the strength of P_katG shows no significant difference under different concentrations of hydrogen peroxide (Fig. 3). In addition, using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria (Fig. 4 & 5), as an inducer still doesn’t induce P_katG.

Fig. 3. Relative fluorescence intensity of P_katG after 4.5-hour incubation with hydrogen peroxide in various concentrations.

Fig. 4. Relative fluorescence intensity of P_katG after 4.5-hour incubation with hydrogen peroxide in low concentrations.

Fig. 5. Relative fluorescence intensity of P_katG after 4.5-hour incubation with hydrogen peroxide in high concentrations.

We compared the relative fluorescence intensity of sfGFP expressed by different promoters under the stimulation by different concentrations of paraquat. As expected, sfGFP expression level of J23100 shows no difference between the groups with and without paraquat. P_soxS with its activator, SoxR (BBa_K37710XX), is highly activated under oxidative stress and has the highest gene expression level compared to J23100, P_katG (BBa_K37710XX), and P_soxS (BBa_K37710XX) solely (Fig. 6).

Fig. 6. Comparison of different promoter strengths by relative fluorescence intensity after 4.5-hour incubation with paraquat in various concentrations.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 6
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 83