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.
See example registry pages for the 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 Since the sample buffer from the Agilent Tapestation 4200 was out of date, with a degraded upper marker, the size of RNA at each peak is not completely accurate.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.
Results from our final tests are shown below.
We mixed our Cas13a, an sgRNA, the corresponding target RNA and our fluorescent probes and saw an increase in fluorescence with time for PLAUR, CXCL8, RGS16 and NR4A1. Tis shows that when activated by the correct sgRNA Cas13a binds to the target and cleaves the probe. However, there was no increase in fluorescence for FOSB. This is because we discovered a mistake in the folding of the FOSB sgRNA. However this proved beneficial as it shows that if the sgRNA does not fold correctly Cas13a cannot cleave the probes.
Conclusion
We successfully expressed our Cas13a enzyme and achieved reasonable purity. We can tell the Cas13a system worked because we got positive results when the Cas13a, sgRNA, target and probe were added together (as shown in the graph above), but also because we had negative controls that caused no reaction. These were:
The system missing one of the 4 parts showing all parts are necessary.
The system with the wrong target showing that the system only activates with the correct RNA sequence (data not shown on this graph please see Measurement section on Wiki).
The system with the spacer FOSB which folded incorrectly, showing the testing for the folding of sequences was necessary.
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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