Difference between revisions of "Part:BBa K5124012"

Revision as of 13:18, 22 August 2024

Cas13a crRNA

Usage and Biology

The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems.

This basic part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type VI CRISPR loci of Leptotrichia wadei [1]. This sequence is combined with one of five spacer sequences that are complimentary to our target bovine RNA. Once transcribed into RNA, the 29-nucleotide repeat sequence folds into a single hairpin loop, which is recognised and bound by LwCas13a, leaving the 20-nucleotide spacer sequence free to bind to the target RNA (Figure 1).

Characterisation

This crRNA sequence was taken from the paper by Kelner et al. [2]. It was synthesised by IDT as part of one of five composite parts (BBa_K5124035 to BBa_K5124040) each containing: a 3’ spacer sequence (BBa_K5124018 to BBa_K5124023), 5’ T7 promoter BBa_I719005 and BioBrick compatible prefix and suffixes. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [3]) carrying an ampicillin selection marker.

Please see composite parts:
K5124035
K5124036
K5124037
K5124038
K5124039
K5124040
for further usage and results.

References

[1] Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, et al. RNA targeting with CRISPR-Cas13. Nature. 2017 Oct 12; 550(7675):280-4.

[2] Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019 Oct; 14(10):2986-3012.

[3] Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct; 19(3):259-68.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K5124012 short</partinfo>
+===Usage and Biology===
+The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems.
-Common to all Cas13a crRNA sequences to put with the spacer
+This basic part codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type VI CRISPR loci of <i>Leptotrichia wadei</i> [1]. This sequence is combined with one of five spacer sequences that are complimentary to our target bovine RNA. Once transcribed into RNA, the 29-nucleotide repeat sequence folds into a single hairpin loop, which is recognised and bound by LwCas13a, leaving the 20-nucleotide spacer sequence free to bind to the target RNA (Figure 1).
-<!-- Add more about the biology of this part here
+===Characterisation===
-===Usage and Biology===
+This crRNA sequence was taken from the paper by Kelner <i>et al.</i> [2]. It was synthesised by IDT as part of one of five composite parts (BBa_K5124035 to BBa_K5124040) each containing: a 3’ spacer sequence (BBa_K5124018 to BBa_K5124023), 5’ T7 promoter [https://parts.igem.org/Part:BBa_I719005 BBa_I719005] and BioBrick compatible prefix and suffixes. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [3]) carrying an ampicillin selection marker.
+Please see composite parts:
+<br>[https://parts.igem.org/Part:BBa_K5124035 K5124035]
+<br>[https://parts.igem.org/Part:BBa_K5124036 K5124036]
+<br>[https://parts.igem.org/Part:BBa_K5124037 K5124037]
+<br>[https://parts.igem.org/Part:BBa_K5124038 K5124038]
+<br>[https://parts.igem.org/Part:BBa_K5124039 K5124039]
+<br>[https://parts.igem.org/Part:BBa_K5124040 K5124040]
+<br>for further usage and results.
+===References===
+[1] Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, et al. RNA targeting with CRISPR-Cas13. Nature. 2017 Oct 12; 550(7675):280-4.
+[2] Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019 Oct; 14(10):2986-3012.
+[3] Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct; 19(3):259-68.
+===Sequence and Features===
 <!-- -->
-<span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K5124012 SequenceAndFeatures</partinfo>