Part:BBa_K5124003
Cas12a RD4_target_a
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 2007 Taylor et al. demonstrated that it was possible to detect bTB DNA in cattle lymph nodes [2]. One the targets for their assay was RD4 which is known as a region of difference between different subspecies of tuberculosis causing bacteria. Therefore, the RD4 region has the potential to be used to specifically detect bTB.
This basic part is a target sequence; a 120-nucleotide DNA sequence from the Mycobacterium bovis AF2122/97 genome (Accession number LT70834) [3]. The bottom strand contains a 20-nucleotide region that is complimentary to the spacer sequence RD4_a (see basic part BBa_K5124015 for further details). This target sequence was used in our Cas12a 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 with Type IIs compatible prefix and suffixes. As we needed the DNA sequence be identical to the bTB genome, we were unable to remove the EcoRI restriction enzyme site. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [4]) carrying an ampicillin selection marker.
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. Garnier T, Eiglmeier K, Camus JC, Medina N, Mansoor H, Pryor M, et al. The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U S A. 2003 Jun 24; 100(13):7877-82.
4. 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 EcoRI site found at 76
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 76
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 76
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 76
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 76
- 1000COMPATIBLE WITH RFC[1000]
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