Difference between revisions of "Part:BBa K2611001"
 
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<partinfo>BBa_K2611001 short</partinfo>
 
<partinfo>BBa_K2611001 short</partinfo>
  
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We added a spacer sequence and NGG site closely before the promoter (BBa_B0034). This spacer sequence can be recognized by the sgRNA we designed to direct dCas9. Once dCas9 binds with the spacer sequence, the expression of GFP will be repressed.  
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We added a spacer sequence and NGG site closely before the promoter J23101 of part:[https://parts.igem.org/Part:BBa_J364000 BBa_J364000]. This spacer sequence can be recognized by the sgRNA we designed to direct dCas9. Once dCas9 binds with the spacer sequence, the expression of GFP will be repressed. To be used in pSB1C3.
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To be used in pSB1C3.
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We selected part J23101-GFP (BBa_J364000) as a reporter to test the function of the modified promoter and measure the repression level. The modified promoter does not influence the function of GFP gene as the GFP fluorescence intensity of the modified part is even stronger than the original one (Figure 3).  
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We selected part GFP as a reporter to test the function of the modified promoter and measure the repression level. The modified promoter does not influence the function of GFP gene as the GFP fluorescence intensity of the modified part is even stronger than the original one (Figure 3).
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As for the repression of dCas9 to the GFP expression, we did an experiment to test the repression level (Figure.4). The expression of GFP was repressed obvious according to the Figure 3 and Figure 4.
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So, in '''conclusion''':
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'''1.''' The modification to the promoter J23101 does not repress the expression of the GFP, but increase it.
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'''2.''' The dCas9 binding can obviously repress the expression of GFP. It means the spacer sequence can work well.
  
  
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[[File:SCU_China-2018_spacer_J23101_GFP(3).png]]
 
[[File:SCU_China-2018_spacer_J23101_GFP(3).png]]
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[[File:SCU_China-2018_spacer_J23101_GFP(4).png]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 09:48, 17 October 2018


spacer J23101-GFP

We added a spacer sequence and NGG site closely before the promoter J23101 of part:BBa_J364000. This spacer sequence can be recognized by the sgRNA we designed to direct dCas9. Once dCas9 binds with the spacer sequence, the expression of GFP will be repressed. To be used in pSB1C3.

We selected part GFP as a reporter to test the function of the modified promoter and measure the repression level. The modified promoter does not influence the function of GFP gene as the GFP fluorescence intensity of the modified part is even stronger than the original one (Figure 3).

As for the repression of dCas9 to the GFP expression, we did an experiment to test the repression level (Figure.4). The expression of GFP was repressed obvious according to the Figure 3 and Figure 4.


So, in conclusion:

1. The modification to the promoter J23101 does not repress the expression of the GFP, but increase it.

2. The dCas9 binding can obviously repress the expression of GFP. It means the spacer sequence can work well.


SCU China-2018 spacer J23101 GFP.png

SCU China-2018 spacer J23101 GFP(2).png

SCU China-2018 spacer J23101 GFP(3).png

SCU China-2018 spacer J23101 GFP(4).png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 30
    Illegal NheI site found at 53
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 728