Part:BBa_K2611001

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.

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