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Part:BBa_K4907115
I0500-B0034-vsw-3 rnapN-npuN-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.
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/i0500-b0034-vsw-3-rnapn-npun-b0015.png)
Characterization
Agarose gel electrophoresis (AGE)
When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-3427 bp (lane K4907115).
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/115.png)
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. 10). 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.
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/vsw-3-rnap/fig10.png)
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).
4.G. Qing et al., Cold-shock induced high-yield protein production in Escherichia coli. Nature Biotechnology 22, 877-882 (2004).
5.B. Wang, R. I. Kitney, N. Joly, M. Buck, Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nature Communications 2, 508 (2011).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2687
Illegal BglII site found at 2924
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 1926 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1449
Illegal BsaI.rc site found at 2680
Illegal SapI site found at 961
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