Difference between revisions of "Part:BBa K3982006"
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| − | Cas14a1 is a type V-F exceptionally compact RNA-guided nuclease. Cas14 forms a complex with RNA-guided nuclease and it can cleave ssDNA without PAM specificity. It | + | Cas14a1 is a type V-F exceptionally compact RNA-guided nuclease. Cas14 forms a complex with RNA-guided nuclease and it can cleave ssDNA without PAM specificity. It is tagged with 6xHis and has the Tobacco Etch Virus (TEV) protease and cleavage site for easy purification. In our project CODE M, Cas14a1 and sgRNA complex is used to detect SNPs in the Mycobacterium tuberculosis genome from a sputum sample. Upon detection, it triggers cleavage of a Fluorescence-Quencher pair ssDNA reporter and thus fluorescence is emitted which confirms the presence of SNPs in the genome. |
| − | [ | + | Check the wikipage of [https://2021.igem.org/Team:IISER_Berhampur Team IISER_Berhampur 2021] to know more about the working of this part. |
| + | ===Usage and Biology=== | ||
| + | Cas14a1 (also called as Un1Cas12f1) is a miniature cas protein used in CRISPR/Cas technology identified from the immune system against viral genome in archaebacteria and bacteria. It is a RNA guided nuclease which cleaves ssDNA without any Protospacer Adjacent Motif (PAM) specificity i.e. PAM independent ssDNA cleavage. This enables high-fidelity Single-nucleotide polymorphism (SNP) genotyping. It is guided by a single guide RNA (sgRNA) which has two components - CRISPR RNA (crRNA) and Trans-activating crispr RNA (tracrRNA). | ||
| + | We have used parts [https://parts.igem.org/Part:BBa_K3982001 BBa_K3982001], [https://parts.igem.org/Part:BBa_K3982002 BBa_K3982002], [https://parts.igem.org/Part:BBa_K3982003 BBa_K3982003] to build this composite part. | ||
| + | [[File:Cas14a1_gene_with_6xHisTag_and_TEV_site_cloned_in_pSB1C3.jpeg|700px|thumb|center|6xHis tagged Cas14a1 gene with TEV site]] | ||
| + | === Methodology === | ||
| + | To remove the 6x His Tag, TEV protease and Cas14a1 gene, we propose to insert RE sites Xba1 and Spe1 in Addgene #112502. We propose to purify the recombinant Cas14a1 protein (Part [https://parts.igem.org/Part:BBa_K3982001 BBa_K3982001]) from this BioBrick with the help of 6x His Tag (Part [https://parts.igem.org/Part:BBa_K3982002 BBa_K3982002]) by using immobilized metal affinity chromatography (IMAC). The TEV protease (Part [https://parts.igem.org/Part:BBa_K3982003 BBa_K3982003]) will be used for removal of the 6x His tag after purification of Cas14a1 by cleaving between Glutamine and Serine residues in its sequence. | ||
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Latest revision as of 14:36, 21 October 2021
6xHis - Tagged Cas14a1 with TEV site
Cas14a1 is a type V-F exceptionally compact RNA-guided nuclease. Cas14 forms a complex with RNA-guided nuclease and it can cleave ssDNA without PAM specificity. It is tagged with 6xHis and has the Tobacco Etch Virus (TEV) protease and cleavage site for easy purification. In our project CODE M, Cas14a1 and sgRNA complex is used to detect SNPs in the Mycobacterium tuberculosis genome from a sputum sample. Upon detection, it triggers cleavage of a Fluorescence-Quencher pair ssDNA reporter and thus fluorescence is emitted which confirms the presence of SNPs in the genome.
Check the wikipage of Team IISER_Berhampur 2021 to know more about the working of this part.
Usage and Biology
Cas14a1 (also called as Un1Cas12f1) is a miniature cas protein used in CRISPR/Cas technology identified from the immune system against viral genome in archaebacteria and bacteria. It is a RNA guided nuclease which cleaves ssDNA without any Protospacer Adjacent Motif (PAM) specificity i.e. PAM independent ssDNA cleavage. This enables high-fidelity Single-nucleotide polymorphism (SNP) genotyping. It is guided by a single guide RNA (sgRNA) which has two components - CRISPR RNA (crRNA) and Trans-activating crispr RNA (tracrRNA).
We have used parts BBa_K3982001, BBa_K3982002, BBa_K3982003 to build this composite part.
Methodology
To remove the 6x His Tag, TEV protease and Cas14a1 gene, we propose to insert RE sites Xba1 and Spe1 in Addgene #112502. We propose to purify the recombinant Cas14a1 protein (Part BBa_K3982001) from this BioBrick with the help of 6x His Tag (Part BBa_K3982002) by using immobilized metal affinity chromatography (IMAC). The TEV protease (Part BBa_K3982003) will be used for removal of the 6x His tag after purification of Cas14a1 by cleaving between Glutamine and Serine residues in its sequence.
Avoid using EcoR1 and Pst1 RE sites.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1411
Illegal PstI site found at 201
Illegal PstI site found at 367 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1411
Illegal PstI site found at 201
Illegal PstI site found at 367 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1411
Illegal BamHI site found at 1005 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1411
Illegal PstI site found at 201
Illegal PstI site found at 367 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1411
Illegal PstI site found at 201
Illegal PstI site found at 367
Illegal AgeI site found at 661 - 1000COMPATIBLE WITH RFC[1000]