Difference between revisions of "Part:BBa K5124041"

Revision as of 20:21, 28 September 2024

T7 promoter for in-vitro transcription

Usage and Biology

In-vitro transcription allows for synthesis of RNA from DNA in a cell free system.

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. To develop a test for RNA we needed a safe method to produce the RNA we needed for our tests. By synthesising short segments of DNA and using in-vitro transcription to produce RNA we removed the possibility of either using or producing any toxic components.

The bacteriophage T7 promoter is a commonly used promoter for protein expression. It is recognised by the T7 RNA polymerase and in the absence of any other control elements is constitutive. 24 sequences that include ‘T7’ in the description are listed in the Registry of Standard Biological Parts catalogue of promoters (e.g. BBa_R0085 and BBa_J64997) and there are many more versions on the registry that are not in the catalogue. Unfortunately, we could not find any that had the 3’ sequence that we needed for in-vitro transcription.

Berckert and Masquida reported that most T7 promoters will produce RNA transcripts with G nucleotides at positions +1, +2 and +3, with the first two being critical for transcriptional yield [3]. In the 2019 paper where the CRISPR-Cas13a SHERLOCK detection protocol was published, Kelner et al [4] designed their in-vitro transcription reactions to include the T7-3G IVT primer (Figure 1) which added the 3rd G to the end of the 3’ end of the T7 promoter sequence. We therefore wanted to use this sequence to drive transcription of our Cas12a and Cas13a sgRNA sequences and our Cas13a target RNA sequences.

See example registry pages for the results. BBa_K5124035

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;3:12.

3. Beckert B, Masquida B. Synthesis of RNA by in vitro transcription. Methods Mol Biol. 2011;703:29-41.

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

Sequence and Features