Difference between revisions of "Part:BBa K3365009"
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===Usage and Biology===
 
===Usage and Biology===
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This part is used to combine the signal of arabinose and the off-target or not of dCas9. The uninduced transcriptional level downstream the signaling is very low. In the presence of arabinose, transcription from the pBAD promoter is turned on. In the presence of both arabinose and the complex of dCas9 and sgRNA, the complex might bind to the lure3 sequence and the transcription might be partially inhibited because of the potential block of RNAP.
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===Rerults===
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The lure3 sequence is added to the promoter of the L-arabinose operon of E. coli (pBAD) (BBa_K3365003) by PCR. The eletrophoretic profile of the PCR product and the sequencing result reveal the successful construction of the fragment.
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<center>[[File:BBa_K3365009_Eletrophoretic_profile.png]]</center>
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<center><b>Figure2.</b> Eletrophoretic profile of the PCR product</center>
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<center>Lane 5-8: 8PCR product</center>
  
 
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Revision as of 19:15, 27 October 2020


Lure3 sequence downstream of pBAD

The PAM and potential off-target sequence are located directly downstream of the promoter (BBa_K3365003), where dCas9 might bind wrongly and block RNAP. In our part, the “lure3” is the potential off-target sequence for PDCD1 CRISPR gene editing predicted by our off-target predictor software built in 2019, which might be identified and bound by the complex of dCas9 and sgRNA. According to the off-target predictor software, the relative off-target rate is 73.6673%.

BBa K3365009.png
Figure1. Gene circuit


Usage and Biology

This part is used to combine the signal of arabinose and the off-target or not of dCas9. The uninduced transcriptional level downstream the signaling is very low. In the presence of arabinose, transcription from the pBAD promoter is turned on. In the presence of both arabinose and the complex of dCas9 and sgRNA, the complex might bind to the lure3 sequence and the transcription might be partially inhibited because of the potential block of RNAP.

Rerults

The lure3 sequence is added to the promoter of the L-arabinose operon of E. coli (pBAD) (BBa_K3365003) by PCR. The eletrophoretic profile of the PCR product and the sequencing result reveal the successful construction of the fragment.

BBa K3365009 Eletrophoretic profile.png
Figure2. Eletrophoretic profile of the PCR product
Lane 5-8: 8PCR product

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 74
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 56