Difference between revisions of "Part:BBa K4907111"

 
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===Usage and Biology===
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===Biology===
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====pVSW-3(16)====
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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 psychrophilic 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(16) is one of them.
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===Usage and Design===
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In order to construct a matching expression system of the VSW-3 RNAP, we characterized its potentially useful promoters using RFP (<partinfo>BBa_K4907037 </partinfo>) as the reporter. pVSW-3(16) is one of the more efficient promoters in the series. Different sub parts were assembled into pSB3K3 plasmid backbone to get the composite part <partinfo>BBa_K4907110</partinfo> (Fig. 1). The plasmid was transformed into <i>E. coli</i> DH5α and the positive transformants were confirmed by kanamycin, colony PCR and sequencing.
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===Characterization===
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====Agarose gel electrophoresis (AGE)====
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When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1220bp (lane K4907110).
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/bba-k4907110-p.png" width="400px"></html></center>
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<center><html><B>Fig. 2 The result of colony PCR. Plasmid BBa_K4907110_pSB3K3 </B></html></center>
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====Comparison of series promoters from pVSW-3(19) to pVSW-3(16)====
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The regulatory plasmid containing VSW-3 RNAP and the expressive plasmids with different promoters were transformed into <i>E. coli</i> BL21(DE3). The correct dual-plasmid system was confirmed by chloramphenicol and kanamycin. We characterized the series promoters from pVSW-3(19) to pVSW-3(16) using RFP under 25 ℃. As shown in Fig. 3, pVSW-3(19), pVSW-3(18), and pVSW-3(16) showed better than pVSW-3(16).
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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 16:25, 10 October 2023


pVSW-3(16)-B0034-rfp-B0015

1

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 472
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 594
  • 1000
    COMPATIBLE WITH RFC[1000]