Part:BBa_K4907018

vsw-3 rnapN-npuN

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.

Fig. 1 Gene circuit of BBa K4907115

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).

Fig. 2 The result of colony PCR. Plasmid BBa_K4907115_pSB1C3

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.

Fig. 3 Characterizations for testing the activity of different forms of VSW-3 RNAP at 25 °C in BL21(DE3). p-value: no significance (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****).

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1451
  Illegal BglII site found at 1688
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 690
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 213
  Illegal BsaI.rc site found at 1444