Difference between revisions of "Part:BBa K4907111"
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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_K4907111</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. | 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_K4907111</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. | ||
| − | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/ | + | <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 pVSW-3 series promoter reporting circuit </B></html></center> | <center><html><B>Fig. 1 Gene circuit of pVSW-3 series promoter reporting circuit </B></html></center> | ||
Revision as of 10:51, 12 October 2023
pVSW-3(16)-B0034-rfp-B0015
Biology
pVSW-3(16)
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 et al. characterized pVSW-3 series promoters for the first time and pVSW-3(16) is one of them.
Usage and Design
In order to construct a matching expression system of the VSW-3 RNAP, we characterized its potentially useful promoters using RFP (BBa_K4907037) 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 BBa_K4907111 (Fig. 1). The plasmid was transformed into E. coli DH5α and the positive transformants were confirmed by kanamycin, colony PCR, and sequencing.
Characterization
Agarose gel electrophoresis (AGE)
When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1197 bp (lane K4907111).
Comparison of series promoters from pVSW-3(19) to pVSW-3(16)
The regulatory plasmid containing VSW-3 RNAP and the expressive plasmids with different promoters were transformed into E. coli 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(17) showed better than pVSW-3(16).
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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 472
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 594
- 1000COMPATIBLE WITH RFC[1000]