Part:BBa_K3771047

PkatG

Description

P_katG is the promoter of the katG gene. KatG protein has the function of bifunctional hydroperoxidase I (HPI) and plays an important role in oxidative stress response. The katG promoter (P_katG) is stimulated by oxidative stress, leading to the expression of the downstream gene. Besides, P_katG is activated by OxyR protein^[1].

Fig. 1. Transcription unit of the katG gene in E. coli K-12^[2].

Usage and Biology

After conducting colony PCR from the E. coli MG1655, the P_katG fragment can be amplified from the chromosome and the experiment result can be checked by agarose gel electrophoresis. The result is shown in Fig. 2. The part has been confirmed by sequencing and has no mutations.

Fig. 2. The electrophoresis result of P_katG fragment from the chromosome PCR. M: Marker; Lane 1: katG promoter (P_katG) (217 bp).

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 paraquat in low concentrations.

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

References

1. Italiani VCS, Silva Neto JF, Braz VS, Marques MV. Regulation of Catalase-Peroxidase KatG Is OxyR Dependent and Fur Independent in Caulobacter crescentus. Journal of Bacteriology. 2011;193(7):1734-1744. doi:10.1128/jb.01339-10

2. Keseler IM, Mackie A, Santos-Zavaleta A, et al. The EcoCyc database: reflecting new knowledge about Escherichia coli K-12. Nucleic Acids Res. 2017;45(D1):D543-D550. doi:10.1093/nar/gkw1003

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
  COMPATIBLE WITH RFC[1000]