Part:BBa_K5124041
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.
Design and Characterisation
Due to the minimum synthesis length of 125 base pairs for IDT gBlocks, this basic part was synthesised as part of a composite part. sgRNA composite parts contained the promoter with a crRNA sequence for either LbCas12a or LwCas13a and a 3’ spacer sequence from either the cow genome or bTB genome respectively. Target RNA composite parts contained the promoter with a target sequence from the cow genome. All composite parts were flanked by Type IIS compatible prefix and suffixes. The gBlock was cloned into a high copy plasmid (origin of replication from pUC18 (3)) carrying an ampicillin selection marker.
See example registry pages for the final results. BBa_K5124035
Results
Results from in-vitro transcription reactions are shown below.
All columns on the RNA tape showed there was RNA present at around the expected length for sgRNA, and target RNA. Since the sample buffer from the Agilent Tapestation 4200 was out of date, with a degraded upper marker, the size of RNA at each peak is not completely accurate. But, consistently there are strong peaks within the expected range. There are also signs of transcribed RNA, from plasmid templates that did not cleave properly, which terminated further through the pX1800 plasmid at a terminator. There is also signs of environmental contamination in all but one Cas13a target’s, however this was not expected to effect results.
Conclusion
These results of successful transcription (and on other pages those transcribed parts also being used for a successful Cas13a system) show that this specific T7 promoter was a useful tool for both our team to use, but also for future teams to use if they needed the GGG 3' end on their promoter, or if they also wanted to transcribe sgRNA to bind to Cas13a or Cas12a.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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