Difference between revisions of "Part:BBa K3365008"
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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%. | 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%. | ||
| − | [[File:Gene_circuit_2.png]] | + | <center>[[File:Gene_circuit_2.png]]</center> |
| − | <b>Figure1.</b> Gene circuit | + | <center><b>Figure1.</b> Gene circuit</center> |
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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. | 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. | ||
| − | [[File:Eletrophoretic_profile_of_the_PCR_result_02.png]] | + | <center>[[File:Eletrophoretic_profile_of_the_PCR_result_02.png]]</center> |
| − | <b>Figure2.</b> Eletrophoretic profile of the PCR | + | <center><b>Figure2.</b> Eletrophoretic profile of the PCR product</center> |
| + | <center>Lane 1-4: PCR product</center> | ||
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Latest revision as of 19:05, 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%.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 74
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 56