Difference between revisions of "Part:BBa K3771047"
 
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<p align="center">Fig. 1. Transcription unit of the <i>katG</i> gene in <i>E. coli</i> K-12<sup>[2]</sup>.</p>
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<p align="center">Fig.1. Transcription unit of the <i>katG</i> gene in <i>E. coli</i> K-12<sup>[2]</sup>.</p>
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<br>After conducting colony PCR from the <i>E. coli</i> MG1655, the <i>P<sub>katG</sub></i> 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.
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<br>After conducting colony PCR from the <i>E. coli</i> MG1655, the <i>P<sub>katG</sub></i> 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.
 
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<img src="https://2021.igem.org/wiki/images/d/d3/T--NCKU_Tainan--PkatG_PCR.png"​ style="width:35%;">
 
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<p align="center">Fig.2. The electrophoresis result of <i>P<sub>katG</sub></i> fragment from the chromosome PCR. M: Marker; Lane 1:  katG promoter (<i>P<sub>katG</sub></i>) (217 bp).
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<p align="center">Fig. 2. The electrophoresis result of <i>P<sub>katG</sub></i> fragment from the chromosome PCR. M: Marker; Lane 1:  katG promoter (<i>P<sub>katG</sub></i>) (217 bp).
 
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<br>The promoter strength of <i>P<sub>katG</sub></i> is determined by the expression level of the reporter, sfGFP, under oxidative stress. While using hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) as the inducer, the strength of <i>P<sub>katG</sub></i> 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 <i>P<sub>katG</sub></i>.
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<br>The promoter strength of <i>P<sub>katG</sub></i> is determined by the expression level of the reporter, sfGFP, under oxidative stress. While using hydrogen peroxide (H2O2) as the inducer, the strength of <i>P<sub>katG</sub></i> 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 <i>P<sub>katG</sub></i>.
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<img src="https://static.igem.org/mediawiki/parts/2/29/T--NCKU_Tainan--Del_High_sfGFP_Expression_%28PkatG%29.png"​ style="width:45%;">
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<p align="center">Fig.3. Relative fluorescence intensity of <i>P<sub>katG</sub></i> after 4.5-hour incubation with hydrogen peroxide in various concentrations.
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<p align="center">Fig. 3. Relative fluorescence intensity of <i>P<sub>katG</sub></i> after 4.5-hour incubation with hydrogen peroxide in various concentrations.
 
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<img src="https://static.igem.org/mediawiki/parts/5/52/T--NCKU_Tainan--Del_Low_sfGFP_Expression_%28PkatG%29.png"​ style="width:45%;">
 
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<p align="center">Fig.4. Relative fluorescence intensity of <i>P<sub>katG</sub></i> after 4.5-hour incubation with paraquat in low concentrations.
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<p align="center">Fig. 4. Relative fluorescence intensity of <i>P<sub>katG</sub></i> after 4.5-hour incubation with paraquat in low concentrations.
 
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<img src="https://static.igem.org/mediawiki/parts/2/29/T--NCKU_Tainan--Del_High_sfGFP_Expression_%28PkatG%29.png"​ style="width:45%;">
 
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<p align="center">Fig.5. Relative fluorescence intensity of <i>P<sub>katG</sub></i> after 4.5-hour incubation with paraquat in high concentrations.
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<p align="center">Fig. 5. Relative fluorescence intensity of <i>P<sub>katG</sub></i> after 4.5-hour incubation with paraquat in high concentrations.
 
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<br><b style="font-size:1.3rem">References</b>
 
<br><b style="font-size:1.3rem">References</b>
 
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<br>1. ItalianiVCS, Silva NetoJF, BrazVS, 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
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<br>1. Italiani VCS, Silva Neto JF, Braz VS, Marques MV. Regulation of Catalase-Peroxidase KatG Is OxyR Dependent and Fur Independent in Caulobacter crescentus. <i>Journal of Bacteriology</i>. 2011;193(7):1734-1744. doi:10.1128/jb.01339-10
 
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<br>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
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<br>2. Keseler IM, Mackie A, Santos-Zavaleta A, et al. The EcoCyc database: reflecting new knowledge about Escherichia coli K-12. <i>Nucleic Acids Res</i>. 2017;45(D1):D543-D550. doi:10.1093/nar/gkw1003
 
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Latest revision as of 02:31, 20 October 2021


PkatG


Description


PkatG 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 (PkatG) is stimulated by oxidative stress, leading to the expression of the downstream gene. Besides, PkatG 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 PkatG 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 PkatG fragment from the chromosome PCR. M: Marker; Lane 1: katG promoter (PkatG) (217 bp).


The promoter strength of PkatG is determined by the expression level of the reporter, sfGFP, under oxidative stress. While using hydrogen peroxide (H2O2) as the inducer, the strength of PkatG 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 PkatG.


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


Fig. 4. Relative fluorescence intensity of PkatG after 4.5-hour incubation with paraquat in low concentrations.


Fig. 5. Relative fluorescence intensity of PkatG 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]