Difference between revisions of "Part:BBa K4907018"
Line 3: | Line 3: | ||
<partinfo>BBa_K4907018 short</partinfo> | <partinfo>BBa_K4907018 short</partinfo> | ||
− | 1 | + | ===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 <i><b>in vitro</b></i> in 2022. VSW-3 RNAP showed a good low-temperature performance, producing fewer terminal and full-length dsRNA byproducts than the T7 RNAP transcript <i>in vitro</i> (1). Moreover, the <i>in vitro</i> transcription products of VSW-3 RNAP were used to prepare mRNA for mRNA therapy <i>in vivo</i> 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. | ||
+ | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/i0500-b0034-vsw-3-rnapn-npun-b0015.png" width="400px"></html></center> | ||
+ | <center><html><B>Fig. 1 Gene circuit of BBa K4907115 </B></html></center> | ||
+ | |||
+ | ===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). | ||
+ | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/115.png" width="400px"></html></center> | ||
+ | <center><html><B>Fig. 2 The result of colony PCR. Plasmid BBa_K4907115_pSB1C3 </B></html></center> | ||
+ | |||
+ | ====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 <i>L</i>-arabinose induced promoter BBa_I0500 then constructed the expressing circuit, <partinfo>BBa_K4907115</partinfo> and <partinfo>BBa_K4907116</partinfo> on the backbone pSB1C3. The VSW-3 RNAP-expressing plasmid (<partinfo>BBa_K4907114</partinfo>_pSB1C3), and the split halves-expressing plasmids or the control (BBa_I0500) were co-transformed with the pVSW-3(18) reporting circuit (<partinfo>BBa_K4907108</partinfo>) 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 <b>the single half of the split RNA polymerase cannot function to trigger the expression of pVSW-3(18) promoter.</b> | ||
+ | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/vsw-3-rnap/fig10.png" width="300px"></html></center> | ||
+ | <center><b>Fig. 3 Characterizations for testing the activity of different forms of VSW-3 RNAP at 25 °C in BL21(DE3).</b> <i>p</i>-value: no significance (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****).</center> | ||
+ | |||
+ | ===Reference=== | ||
+ | 1. H. Xia et al., Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription. <i>RNA Biology</i> <b>19</b>, 1130-1142 (2022). | ||
+ | |||
+ | 2.G. Wang et al., mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation. <i>Cell Insight</i> <b>1</b>, 100056 (2022). | ||
+ | |||
+ | 3.L. Saleh, F. B. Perler, Protein splicing in cis and in trans. <i>Chem Rec</i> <b>6</b>, 183-193 (2006). | ||
+ | |||
+ | 4.G. Qing et al., Cold-shock induced high-yield protein production in Escherichia coli. <i>Nature Biotechnology</i> <b>22</b>, 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. <i>Nature Communications</i> <b>2</b>, 508 (2011). | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Revision as of 12:24, 12 October 2023
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.
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/i0500-b0034-vsw-3-rnapn-npun-b0015.png)
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).
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/115.png)
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.
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/vsw-3-rnap/fig10.png)
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1451
Illegal BglII site found at 1688 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 690
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 213
Illegal BsaI.rc site found at 1444