Difference between revisions of "Part:BBa K4907108"

 
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<partinfo>BBa_K4907108 short</partinfo>
 
<partinfo>BBa_K4907108 short</partinfo>
  
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===Biology===
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====pVSW-3(18)====
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Some RNA polymerases of eukaryotes and viruses have domains that specifically recognize DNA base sequences, and they 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.
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===Usage and design===
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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).
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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 <r>E. coli</r>.
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<center><html><imgsrc="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/109-1.png" width="400px"></html></center>
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<center><html><B>Fig. 1 Gene circuit of <partinfo>BBa_K4907122</partinfo>_pSB3K3 </B></html></center>
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===Characterization===
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====Agarose gel electrophoresis (AGE)====
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When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1220bp (lane K4907109).
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<center><html><imgsrc="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/109-1.png" width="400px"></html></center>
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<center><html><B> Fig. 2 The result of colony PCR. Plasmid BBa_K4907109_pSB3K3 </B></html></center>
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====Comparison of series promoters: pVSW-3(GGG), pVSW-3(genome)====
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In order to find a promoter that can function efficiently in Escherichia coli, 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.
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<center><html><imgsrc="hhttps://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/ggg-gemone.png" width="400px"></html></center>
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<center><html><B> Fig. 3 The comparison of normalized fluorescence intensity the promoters pVSW-3(18), pVSW-3(GGG) and pVSW-3(genome)</B></html></center>
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===Reference===
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1.S. Borukhov, E. Nudler, RNA polymerase: the vehicle of transcription. <i>Trends in Microbiology</i> <b>16</b>, 126-134 (2008).
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2. H. Xia <i>et al.</i>, 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).
  
 
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Revision as of 17:37, 10 October 2023


pVSW-3 (18)-B0034-rfp-B0015

Biology

pVSW-3(18)

Some RNA polymerases of eukaryotes and viruses have domains that specifically recognize DNA base sequences, and they 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 <r>E. coli</r>.

Fig. 1 Gene circuit of BBa_K4907122_pSB3K3

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).

Fig. 2 The result of colony PCR. Plasmid BBa_K4907109_pSB3K3

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.

Fig. 3 The comparison of normalized fluorescence intensity the promoters pVSW-3(18), pVSW-3(GGG) and pVSW-3(genome)

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 474
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 596
  • 1000
    COMPATIBLE WITH RFC[1000]