Difference between revisions of "Part:BBa K4907115"

Latest revision as of 12:15, 12 October 2023

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.

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).

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.

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