Difference between revisions of "Part:BBa K4907110"
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===Biology=== | ===Biology=== | ||
====pVSW-3(17)==== | ====pVSW-3(17)==== | ||
− | Some RNA polymerases of eukaryotes and viruses have domains that specifically recognize DNA base sequences, and they are specifically matched with their corresponding promoters | + | 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(17) is one of them. |
===Usage and Design=== | ===Usage and Design=== | ||
− | 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(17) 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 E. coli 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(17) 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. |
− | + | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/pvsw-3-all-rfp.png" width="400px"></html></center> | |
− | + | <center><html><B>Fig. 1 Gene circuit of pVSW-3 series promoter reporting circuit </B></html></center> | |
===Characterization=== | ===Characterization=== | ||
====Agarose gel electrophoresis (AGE)==== | ====Agarose gel electrophoresis (AGE)==== | ||
− | When | + | When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1198 bp (lane K4907110). |
− | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/bba- | + | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/bba-k4907110-p.png" width="400px"></html></center> |
− | <center><html><B>Fig. 2 The result of colony PCR. Plasmid | + | <center><html><B>Fig. 2 The result of colony PCR. Plasmid BBa_K4907110_pSB3K3 </B></html></center> |
+ | |||
====Comparison of series promoters from pVSW-3(19) to pVSW-3(16)==== | ====Comparison of series promoters from pVSW-3(19) to pVSW-3(16)==== | ||
− | The regulatory plasmid containing VSW-3 RNAP and the expressive | + | 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(17) showed better than pVSW-3(16). |
+ | <center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/xilieqidongzi19-16.png" width="300px"></html></center> | ||
+ | <center><html><B>Fig. 3 The comparison of normalized fluorescence intensity the series promoters from pVSW-3(19) to pVSW-3(16). </B></html></center> | ||
+ | |||
===Reference=== | ===Reference=== | ||
1. S. Borukhov, E. Nudler, RNA polymerase: the vehicle of transcription. <i>Trends in Microbiology</i> <b>16</b>, 126-134 (2008). | 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 et al., 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). | + | 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). |
Latest revision as of 11:00, 12 October 2023
pVSW-3(17)-B0034-rfp-B0015
Biology
pVSW-3(17)
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(17) 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(17) 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_K4907110 (Fig. 1). The plasmid was transformed into E. coli DH5α and the positive transformants were confirmed by kanamycin, colony PCR and sequencing.
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/pvsw-3-all-rfp.png)
Characterization
Agarose gel electrophoresis (AGE)
When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1198 bp (lane K4907110).
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/bba-k4907110-p.png)
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).
![](https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/xilieqidongzi19-16.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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 473
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 595
- 1000COMPATIBLE WITH RFC[1000]