Part:BBa_K4907117

I0500-B0034-sspC-vsw-3 rnapC-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.

Fig. 1 Gene circuit of BBa_K4907118

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. 2 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 we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-4527bp (lane K4907117).

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

The intien do the trick

Then, the reporting circuit of pVSW-3(18) was co-transformed with BBa_K4907117_pSB1C3 into BL21(DE3) for testing the function of the resulting intact VSW-3 RNAP generated by the junction of the split intein. After induction at 25 °C for 20 hours, the group expressing the split halves of VSW-3 RNAP exhibited a much stronger output signal than that of the control group (BBa_I0500_pSB1C3) (Fig. 11b), proving that the generated intact VSW-3 RNAP also gained its normal function. Despite this, the output signal of this experimental group was not as strong as the native intact one, which might be attributed to the decreasing junction efficiency of the split intein at 25 °C.

Fig. 11 Characterizations of the function of split intein to generate the intact VSW-3 RNAP. a SDS-PAGE analysis of the intact form and split form of VSW-3 RNAP. b Characterizations for testing the regaining function of generated intact VSW-3 RNAP at 25 °C.

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<i>., mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation. <i>Cell Insight 1, 100056 (2022).

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 3787
  Illegal BglII site found at 4024
  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 3026
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2549
  Illegal BsaI.rc site found at 3780
  Illegal SapI site found at 961