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 then turned to investigate the function of split intein and if the junction would result in an intact form of the polymerase with regaining normal function. The characterization circuit (BBa_K4907117) was constructed on the backbone pSB1C3 by placing the SspC-VSW-3 RNAPC and VSW-3 RNAPN-NpuN under the control of L-arabinose induced promoter BBa_I0500 in a bicistronic pattern.

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Each fusion half was placed under the control of cspA promoter (pCspA). In this time, the two pCspA promoters acted as inputs while the pVSW-3(18) promoter played the role of output with the target genes placed downstream. Theoretically, leakage expression will occur at a certain probability for a single pCspA as output, however, when the pVSW-3(18) is set as the output, the leakage at high temperatures will rarely happen due to the low-temperature preference of VSW-3 RNAP even if the leakage of two pCspA promoters occur.

Fig. 9 Structure prediction. a The predicted structure of VSW-3 RNAP. The split site was colored in blue. b The predicted structure of VSW-3 RNAPN-NpuN (BBa_K4907018). The NpuN was colored light pink. c The predicted structure of SspC-VSW-3 RNAPC (BBa_K4907017). The SspC was colored light pink.

Characterization

Agarose gel electrophoresis (AGE)

When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1201 bp (lane K4907130).

<center> Fig. 2 The result of colony PCR. Plasmid BBa_K4907110_pSB3K3

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. 10 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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2687
  Illegal BglII site found at 2924
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 1926
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1449
  Illegal BsaI.rc site found at 2680
  Illegal SapI site found at 961