Difference between revisions of "Part:BBa K4307000"

 
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<partinfo>BBa_K4307000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4307000 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
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<partinfo>BBa_K4307000 parameters</partinfo>
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<h2>Characterization</h2>
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<p>The following figure demonstrates our successful construction.</p>
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<br>
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https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307000-gel.png
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<br>
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      <b>Figure 1: </b> <b>The construction results of OR1-OR2.</b>
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<h3>Fluorescence assay was done to characterize the biobrick. </h3>
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<p>To ensure the normal promoter function of OR1-OR2 and to check whether the promoter could be inhibited by cI protein, we added the gene of EGFP downstream of the promoter (OR1-OR2). The fluorescence results of EGFP protein was compared within three groups: the negative control (the bacteria contain an empty plasmid), the group for the test of promotor function (the bacteria contain plasmid with OR1-OR2-EGFP, but without cI protein) and the group for the test of cI inhibition (the bacteria contain plasmid with OR1-OR2-EGFP and cI protein). It should be mentioned that cI protein located downstream of its natural promoter (OR2-OR3) and was expressed constitutively in bacteria. </p>
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<p>Experiments were performed in <i>E. coli</i> BL21(DE3) cell strain cultured at 37°C or 16°C, which were recorded OD600 and the fluorescence signal after 16 hours of culture. The OD600 was used to normalize the fluorescence signal per cell. In figure 1, reported values represent the average result of two repeated normalised results. </p>
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https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307000.png
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                <b>Figure 2: </b> <b> Fluorescence assay for the verification of OR1-OR2</b>
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<h2>Conclusion</h2>
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<p>Comparing the fluorescence data, it can be found that when we test OR1-OR2 as a promoter alone, a significant fluorescence increment can be observed at 16℃ and 37℃ compared with negative Control. Further comparison of the fluorescence intensity before and after the introduction of cI protein indicates that OR1-OR2 can be significantly inhibited by cI protein.</p>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 15:46, 12 October 2022


OR1-OR2

There are two operators in the genome of bacteriophage, called OR (the right operator) and OL (the left operator) respectively. OR plays an important role in the regulation of gene expression of phage. OR composed of three parts, called OR1, OR2 and OR3. The isolated OR1-OR2 can function as promoter and can be inhibit by cI protein, a repressor expressed by bacteriophage.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]




Characterization

The following figure demonstrates our successful construction.


bba-k4307000-gel.png 

     Figure 1:  The construction results of OR1-OR2.

Fluorescence assay was done to characterize the biobrick.

To ensure the normal promoter function of OR1-OR2 and to check whether the promoter could be inhibited by cI protein, we added the gene of EGFP downstream of the promoter (OR1-OR2). The fluorescence results of EGFP protein was compared within three groups: the negative control (the bacteria contain an empty plasmid), the group for the test of promotor function (the bacteria contain plasmid with OR1-OR2-EGFP, but without cI protein) and the group for the test of cI inhibition (the bacteria contain plasmid with OR1-OR2-EGFP and cI protein). It should be mentioned that cI protein located downstream of its natural promoter (OR2-OR3) and was expressed constitutively in bacteria.

Experiments were performed in E. coli BL21(DE3) cell strain cultured at 37°C or 16°C, which were recorded OD600 and the fluorescence signal after 16 hours of culture. The OD600 was used to normalize the fluorescence signal per cell. In figure 1, reported values represent the average result of two repeated normalised results.



bba-k4307000.png 

               Figure 2:   Fluorescence assay for the verification of OR1-OR2


Conclusion

Comparing the fluorescence data, it can be found that when we test OR1-OR2 as a promoter alone, a significant fluorescence increment can be observed at 16℃ and 37℃ compared with negative Control. Further comparison of the fluorescence intensity before and after the introduction of cI protein indicates that OR1-OR2 can be significantly inhibited by cI protein.