Difference between revisions of "Part:BBa K4907043"
Line 3: | Line 3: | ||
<partinfo>BBa_K4907043 short</partinfo> | <partinfo>BBa_K4907043 short</partinfo> | ||
− | 1 | + | ===Biology=== |
+ | ====pVSW-3(genome)==== | ||
+ | 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(genome) 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(genome) is one of the more efficient promoters in the series. BBa_K4907122_pSB3K3 was constructed as a reporting circuit, for comparing with pVSW-3(GGG) and pVSW-3(18). | ||
+ | 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/pvsw-3-genome-1.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-1168bp (lane K4907122). | ||
+ | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/122-1.png" width="400px"></html></center> | ||
+ | <center><html><B> Fig. 2 The result of colony PCR. Plasmid BBa_K4907122_pSB3K3 </B></html></center> | ||
+ | |||
+ | ====Comparison of series promoters:pSB3K3(pVSW-3(18)), pVSW-3(GGG) and 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(genome), 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 | ||
===Usage and Biology=== | ===Usage and Biology=== |
Latest revision as of 12:22, 12 October 2023
pVSW-3(genome)-RBS
Biology
pVSW-3(genome)
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(genome) 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(genome) is one of the more efficient promoters in the series. BBa_K4907122_pSB3K3 was constructed as a reporting circuit, for comparing with pVSW-3(GGG) and pVSW-3(18). 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.
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/pvsw-3-genome-1.png)
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-1168bp (lane K4907122).
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/122-1.png)
Comparison of series promoters:pSB3K3(pVSW-3(18)), pVSW-3(GGG) and 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.
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/ggg-gemone.png)
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]