Difference between revisions of "Part:BBa K3365008"

 
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<partinfo>BBa_K3365008 short</partinfo>
 
<partinfo>BBa_K3365008 short</partinfo>
  
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Lure2 sequence downstream of pBAD
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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 “lure2” is the potential off-target sequence for <i> PDCD1</i> CRISPR gene editing mentioned in the literature, which might be identified and bound by the complex of dCas9 and sgRNA. According to the reference, the absolute off-target rate is 0.05143%.
  
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[[File:Gene_circuit_2.png]]
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<b>Figure1.</b> Gene circuit
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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 lure2 sequence and the transcription might be partially inhibited because of the potential block of RNAP.
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===Results===
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The lure2 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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[[File:Eletrophoretic_profile_of_the_PCR_result_02.png]]
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<b>Figure2.</b> Eletrophoretic profile of the PCR result
  
 
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Revision as of 13:48, 27 October 2020


Lure2 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 “lure2” is the potential off-target sequence for PDCD1 CRISPR gene editing mentioned in the literature, which might be identified and bound by the complex of dCas9 and sgRNA. According to the reference, the absolute off-target rate is 0.05143%.

Gene circuit 2.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 lure2 sequence and the transcription might be partially inhibited because of the potential block of RNAP.

Results

The lure2 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.

Eletrophoretic profile of the PCR result 02.png

Figure2. Eletrophoretic profile of the PCR result

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