Part:BBa_K4907116
I0500-B0034-sspC-vsw-3 rnapC-B0015
Biology
VSW-3 RNAP
The VSW-3 RNAP is a novel single-subunit RNA polymerase encoded by the chillophilic phage VSW-3, which was first characterized in vitro in 2022. VSW-3 RNAP showed a good low-temperature performance, producing fewer terminal and full-length dsRNA byproducts than the T7 RNAP transcript in vitro (1). Moreover, the in vitro transcription products of VSW-3 RNAP were used to prepare mRNA for mRNA therapy in vivo due to the superior protein expression levels of VSW-3 RNA transcripts, compared to T7 RNAP transcripts (2).
VSW-3 RNAPN-NpuN and SspC VSW-3 RNAPC
Based on the split-intein (3) combined with the novel VSW-3 system. In our design, the VSW-3 RNAP was split into two halves and fused to the split intein SspC and NpuN respectively.
Usage and design
We built BBa K4907115_pSB1C3 and BBa K4907116_pSB1C3 to show that half of the polymerase alone can't function.
Characterization
Agarose gel electrophoresis (AGE)
When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-2763 bp (lane K4907116).
The induction effect of spilt polymerase
For careful verification, we preliminarily tested whether the split form of this VSW-3 RNAP could activate the pVSW-3(18) promoter or not. Each split half was placed under the control of L-arabinose induced promoter BBa_I0500 then constructed the expressing circuit, BBa_K4907115 and BBa_K4907116 on the backbone pSB1C3. The VSW-3 RNAP-expressing plasmid (BBa_K4907114_pSB1C3), and the split halves-expressing plasmids or the control (BBa_I0500) were co-transformed with the pVSW-3(18) reporting circuit (BBa_K4907108) into BL21(DE3), respectively. After induction at 25 °C for 12 h, both the group of VSW-3 RNAPC-NpuN and SspC-VSW-3 RNAPN showed no output signals like the control group, which were much lower than that of the intact VSW-3 RNAP (Fig. 3). Based on this observation, it was convinced that the single half of the split RNA polymerase cannot function to trigger the expression of pVSW-3(18) promoter.
Reference
1. H. Xia et al., Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription. RNA Biology 19, 1130-1142 (2022).
2.G. Wang et al., mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation. Cell Insight 1, 100056 (2022).
3.L. Saleh, F. B. Perler, Protein splicing in cis and in trans. Chem Rec 6, 183-193 (2006).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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