Part:BBa_K5124009
Cas13a PLAUR_target only
Usage and Biology
The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems. The literature suggests that bTB infection in cattle can be detected by nucleic acid biomarkers in both blood [1] and tissue samples [2]. Therefore, there was potential to develop tests looking for both DNA and RNA biomarkers in infected cattle.
In 2021 McLoughlin et al. published RNA-Seq data from cattle infected with bTB at several timepoints during the disease progression [1]. They identified 19 potential biomarkers that were present across the entire length of the infection time course. We would have liked to have tested all 19 sequences but after two rounds of the design-build-test-learn cycle (see our Wiki) we focused on:
CXCL8- chemokine ligand 8, involved in infection response and tissue injury.
FOSB- FBJ murine osteosarcoma viral oncogene homologue B, plays a role in regulating cell proliferation, differentiation and transformation.
NR4A1- nuclear receptor subfamily 4, group A, member 1, plays a role in inflammation and apoptosis.
PLAUR- plasminogen activator, urokinase receptor, a biomarker of inflammation.
RGS16- regulator of G-protein signalling 16, linked to several disease states.
This basic part codes for a target sequence; a 150-nucleotide RNA sequence from the Bos taurus genome (BioProject accession number PRJNA450837). Once transcribed this sequence contains a 24-nucleotide sequence that is complimentary to the spacer sequence PLAUR (see basic part BBa_K5124022 for details). This target sequence was used in our Cas13a assays as a mimic of bTB infected samples from cattle. In the final detection system, this part could be used as a positive control.
This sequence was synthesised by IDT as part of a composite part containing a 5’ T7 promoter (BBa_K5124041) and Type IIs compatible prefix and suffixes. As we needed the RNA sequence be identical to the Bos taurus genome, we were unable to remove the PstI restriction enzyme site. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [3]) carrying an ampicillin selection marker.
Please see composite part BBa_K5124028 for further usage and results.
References
1. McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.
2. Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 112
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 112
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 112
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 112
- 1000COMPATIBLE WITH RFC[1000]
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