Difference between revisions of "Part:BBa K3771091"

 
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<partinfo>BBa_K3771091 short</partinfo>
 
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<br><b style="font-size:1.3rem">Description</b>
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<br>This composite part consists of promoter <i>P<sub>katG</sub></i> (BBa_K3771047) and sfGFP (BBa_K1321337). The <i>katG</i> promoter (<i>P<sub>katG</sub></i>) is stimulated by oxidative stress, leading to the expression of the downstream gene. sfGFP serves as a reporter to evaluate the strength of <i>P<sub>katG</sub></i>.
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===Usage and Biology===
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<br><b style="font-size:1.3rem">Usage and Biology</b>
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<br>This composite part was ligated with the pSAA vector and transformed into <i>E. coli</i>. We conducted colony PCR to verify whether <i>E. coli</i> uptake the correct plasmid. The size of the PCR product was as expected.The part has been confirmed by sequencing and has no mutations.
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<html><div style="width=100%; display:flex; align-items: center; justify-content: center;">
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<img src="https://2021.igem.org/wiki/images/9/94/T--NCKU_Tainan--PkatG-sfGFP_Colony_PCR.png"​ style="width:35%;">
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<p align="center"> Fig.1. (A) The electrophoresis result of colony PCR. M: Marker; Lane 1, 2: pSAA-<i>P<sub>katG</sub></i>-<i>sfgfp</i> (2629 bp); Lane 3: pSAA-J23100-<i>sfgfp</i> (2537 bp). (B) Digested the amplified fragments with HindIII. M: Marker; Lane 1: pSAA-J23100-<i>sfgfp</i>; Lane 2, 3: pSAA-<i>P<sub>katG</sub></i>-<i>sfgfp</i> (147, 278, 377, 1933 bp).
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<br>We conducted a disk assay to examine what kinds of oxidative stress inducers can <i>P<sub>katG</sub></i> 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, <i>P<sub>katG</sub></i> only responds to high-concentration hydrogen peroxide (Figure 2).
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<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>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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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3771050 SequenceAndFeatures</partinfo>
  
 
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Revision as of 15:35, 17 October 2021


PkatG-sfGFP


Description


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



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-PkatG-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-PkatG-sfgfp (147, 278, 377, 1933 bp).


We conducted a disk assay to examine what kinds of oxidative stress inducers can PkatG 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, PkatG only responds to high-concentration hydrogen peroxide (Figure 2).



References

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

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
    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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 73

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