Part:BBa_K5124002
Cas12a EthA_target_b_c
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.
Ethionamide (ETH) is used to treat patients infected with multidrug-resistant TB. One of the enzymes thought to be involved in the activation of the drug in-vivo is the monooxygenase EthA [3]. However, mutations are already being found in TB subspecies, including bTB [4], associated with ethionamide resistant infections. Therefore, this may prove a useful region of the bTB genome to target, in tests designed to look for drug resistant strains.
This basic part is a target sequence; a 122-nucleotide DNA sequence from the Mycobacterium bovis AF2122/97 genome (Accession number LT70834) [5]. The bottom strand contains a 20-nucleotide region that is complimentary to the spacer sequences EthA-b and EthA-c (see basic parts BBa_K5124013 and BBa_K5124014 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 PstI restriction enzyme site. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [6]) 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. Ang ML, Zainul Rahim SZ, Shui G, Dianiskova P, Madacki J, Lin W, et al. An ethA-ethR-deficient Mycobacterium bovis BCG mutant displays increased adherence to mammalian cells and greater persistence in vivo, which correlate with altered mycolic acid composition. Infect Immun. 2014 May; 82(5):1850-9.
4. Borham M, Oreiby A, El-Gedawy A, Hegazy Y, Hemedan A, Al-Gaabary M. Abattoir survey of bovine tuberculosis in tanta, centre of the Nile delta, with in silico analysis of gene mutations and protein-protein interactions of the involved mycobacteria. Transbound Emerg Dis. 2022 Mar; 69(2):434-50.
5. 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.
6. 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 40
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 40
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 40
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 40
Illegal NgoMIV site found at 3 - 1000COMPATIBLE WITH RFC[1000]
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