Part:BBa_K4907114
I0500-B0034-vsw-3 rnap-B0015
Biology
VSW-3 RNAP
VSW-3 RNAP (BBa_K4907016) is a novel single-subunit RNA polymerase encoded by the chillophilic phage VSW-3 from a plateau lakes. The phage's genome was sequenced in 2017. Heng Xia et al. first characterized VSW-3 RNAP in vitro in 2022. In the work of Heng Xia et al., the VSW-3 RNAP showed good low temperature performance, producing fewer terminal and full-length dsRNA byproducts than the T7 RNAP transcript. Further, Wang Guoquan et al. used VSW-3 RNAP for in vitro transcription to prepare mRNA for mRNA therapy, depending on VSW-3 RNA transcripts exhibiting superior protein expression levels compared to T7 RNAP transcripts (1).
Usage and design
To introduce a polymerase with sufficient performance at low temperatures to create our And-logic gate, we found the VSW-3 RNAP. In order to successfully characterize VSW-3 RNAP, we constructed I0500-B0034-VSW-3 RNAP-B0015_PSB1C3 (BBa_K4907114_pSB1C3), and characterized its matched promoter by means of double plasmid transformation. The methods to improve its expression efficiency were also explored. See the vsw-3 rnap page for more information.
Characterization
Agarose gel electrophoresis (AGE)
When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1221bp (lane K4907109).
Comparison of series promoters
From pVSW-3(19) to pVSW-3(16)
The regulatory plasmid containing VSW-3 RNAP and the expressive plasmid with different promoters were transformed into E. coli BL21(DE3). The correct dual plasmid system was confirmed by chloramphenicol and kanamycin. We characterized the series promoters from pVSW-3(19) to pVSW-3(16) using RFP under 25 ℃. As shown in Fig. 3, pVSW-3(19), pVSW-3(18) and pVSW-3(17) showed better than pVSW-3(16).
Fig. 3 The comparison of normalized fluorescence intensity the series promoters from pVSW-3(19) to pVSW-3(16).
pVSW-3(GGG), pVSW-3(genome)
In order to find a promoter that can function efficiently in E. coli, we constructed BBa_K4907109_pSB3K3(pVSW-3(18)) and BBa_K4907112_pSB3K3(pVSW-3(GGG)) to explore the effect of the structure of the 3' terminal of the promoter on its efficiency. The results are shown in the figure, with BBa_K4907109_pSB3K3 showing the highest efficiency.
Fig. 4 The comparison of normalized fluorescence intensity the promoters pVSW-3(18), pVSW-3(GGG) and pVSW-3(genome)
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).
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
Illegal AgeI site found at 1926 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1449
Illegal SapI site found at 961
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