Difference between revisions of "Part:BBa K4907109"
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<partinfo>BBa_K4907109 short</partinfo> | <partinfo>BBa_K4907109 short</partinfo> | ||
− | 1 | + | ===Biology=== |
+ | ====pVSW-3(18)==== | ||
+ | Some RNA polymerases of eukaryotes and viruses have domains that specifically recognize DNA base sequences and are specifically matched with their corresponding promoters (1). VSW-3 RNAP is encoded by the chillophilic phage VSW-3 in plateau lakes and has low-temperature specificity (2). Hengxia <i>et al</i>. characterized pVSW-3 series promoters for the first time and pVSW-3(18) is one of them. | ||
+ | |||
+ | ===Usage and design=== | ||
+ | XMU-China has developed a novel RNA polymerase, VSW-3 RNAP and we characterized its potentially useful promoters in order to construct a matching expression system. pVSW-3(18) is one of the more efficient promoters in the series. BBa_K4907109_pSB3K3 was constructed as a reporting circuit, for comparing with pVSW-3(GGG) and pVSW-3(genome). | ||
+ | By characterizing these three promoters, we hope to determine the effect of the 3'terminal structure of the promoter for VSW-3 RNAP on its efficiency and to identify a VSW-3 expression system that can effectively function in <i>E. coli</i>. | ||
+ | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/pvsw-3-all-gfp.png" width="400px"></html></center> | ||
+ | <center><html><B>Fig. 1 Gene circuit of <partinfo>BBa_K4907109</partinfo>_pSB3K3 and <partinfo>BBa_K4907122</partinfo>_pSB3K3 </B></html></center> | ||
+ | |||
+ | ===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-1220bp (lane K4907109). | ||
+ | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/109-1.png" width="400px"></html></center> | ||
+ | <center><html><B> Fig. 2 The result of colony PCR. Plasmid BBa_K4907109_pSB3K3 </B></html></center> | ||
+ | |||
+ | ====Comparison of series promoters: pVSW-3(GGG), pVSW-3(genome)==== | ||
+ | In order to find a promoter that can function efficiently in <i>Escherichia coli</i>, we constructed <partinfo>BBa_K4907109</partinfo>_pSB3K3(pVSW-3(18)), <partinfo>BBa_K4907112</partinfo>_pSB3K3(pVSW-3(GGG)) and <partinfo>BBa_K4907122</partinfo>_pSB3K3(pVSW-3(genome)) to explore the effect of the structure of the 3' terminal of the promoter on its efficiency. The results are shown in the figure, with <partinfo>BBa_K4907109</partinfo>_pSB3K3 showing the highest efficiency. | ||
+ | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/ggg-gemone.png" width="300px"></html></center> | ||
+ | <center><html><B> Fig. 3 The comparison of normalized fluorescence intensity the promoters pVSW-3(18), pVSW-3(GGG) and pVSW-3(genome).</B> <i>p</i>-value: 0.0021 (**), 0.0002 (***), <0.0001 (****)</html></center> | ||
+ | |||
+ | ===Reference=== | ||
+ | 1. S. Borukhov, E. Nudler, RNA polymerase: the vehicle of transcription. <i>Trends in Microbiology</i> <b>16</b>, 126-134 (2008). | ||
+ | |||
+ | 2. H. Xia <i>et al.</i>, Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in <i>in vitro</i> transcription. <i>RNA Biology</i> <b>19</b>, 1130-1142 (2022). | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Latest revision as of 10:04, 11 October 2023
pVSW-3 (18)-B0034-gfp-B0015
Biology
pVSW-3(18)
Some RNA polymerases of eukaryotes and viruses have domains that specifically recognize DNA base sequences and are specifically matched with their corresponding promoters (1). VSW-3 RNAP is encoded by the chillophilic phage VSW-3 in plateau lakes and has low-temperature specificity (2). Hengxia et al. characterized pVSW-3 series promoters for the first time and pVSW-3(18) is one of them.
Usage and design
XMU-China has developed a novel RNA polymerase, VSW-3 RNAP and we characterized its potentially useful promoters in order to construct a matching expression system. pVSW-3(18) is one of the more efficient promoters in the series. BBa_K4907109_pSB3K3 was constructed as a reporting circuit, for comparing with pVSW-3(GGG) and pVSW-3(genome). By characterizing these three promoters, we hope to determine the effect of the 3'terminal structure of the promoter for VSW-3 RNAP on its efficiency and to identify a VSW-3 expression system that can effectively function in E. coli.
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-1220bp (lane K4907109).
Comparison of series promoters: pVSW-3(GGG), pVSW-3(genome)
In order to find a promoter that can function efficiently in Escherichia coli, we constructed BBa_K4907109_pSB3K3(pVSW-3(18)), BBa_K4907112_pSB3K3(pVSW-3(GGG)) and BBa_K4907122_pSB3K3(pVSW-3(genome)) to explore the effect of the structure of the 3' terminal of the promoter on its efficiency. The results are shown in the figure, with BBa_K4907109_pSB3K3 showing the highest efficiency.
Reference
1. S. Borukhov, E. Nudler, RNA polymerase: the vehicle of transcription. Trends in Microbiology 16, 126-134 (2008).
2. 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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]