Difference between revisions of "Part:BBa K4907110"

(→‎Agarose gel electrophoresis (AGE))
(→‎Comparison of series promoters from pVSW-3(19) to pVSW-3(16))
 
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===Biology===
 
===Biology===
 
====pVSW-3(17)====
 
====pVSW-3(17)====
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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(17) is one of them.
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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(17) is one of them.
 
===Usage and Design===
 
===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(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.
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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(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.
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/pvsw-3-all-rfp.png" width="400px"></html></center>
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<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)====
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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 K4907110).
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When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1198 bp (lane K4907110).
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/bba-k4907111-p16-4.png" width="400px"></html></center>
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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>
 
<center><html><B>Fig. 2 The result of colony PCR. Plasmid BBa_K4907110_pSB3K3 </B></html></center>
 
<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)====
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The regulatory plasmid containing VSW-3 RNAP and the expressive plasmid 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).
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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(17) showed better than pVSW-3(16).
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/xilieqidongzi19-16.png" width="300px"></html></center>
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<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>
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===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).
  
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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).
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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 <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.

Fig. 1 Gene circuit of pVSW-3 series promoter reporting circuit

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).

Fig. 2 The result of colony PCR. Plasmid BBa_K4907110_pSB3K3

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).

Fig. 3 The comparison of normalized fluorescence intensity the series promoters from pVSW-3(19) to 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 473
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 595
  • 1000
    COMPATIBLE WITH RFC[1000]