Composite

Part:BBa_K4907108

Designed by: Yannan You   Group: iGEM23_XMU-China   (2023-09-15)
Revision as of 17:40, 10 October 2023 by CspA (Talk | contribs) (→‎Usage and design)


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]


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