Difference between revisions of "Part:BBa K4907108"

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===Biology===
 
===Biology===
 
====pVSW-3(18)====
 
====pVSW-3(18)====
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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(18) 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(18) is one of them.
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===Usage and Design===
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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(18) 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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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).
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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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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 <i>E. coli</i>.
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<center><html><B>Fig. 1 Gene circuit of pVSW-3 series promoter reporting circuit </B></html></center>
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<center><html><imgsrc="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/109-1.png" width="400px"></html></center>
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<center><html><B>Fig. 1 Gene circuit of <partinfo>BBa_K4907122</partinfo>_pSB3K3 </B></html></center>
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===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 K4907109).
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When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1199 bp (lane K4907108).
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<center><html><imgsrc="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/109-1.png" width="400px"></html></center>
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/108-1.png" width="400px"></html></center>
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<center><html><B> Fig. 2 The result of colony PCR. Plasmid BBa_K4907109_pSB3K3 </B></html></center>
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<center><html><B>Fig. 2 The result of colony PCR. Plasmid BBa_K4907108_pSB3K3 </B></html></center>
  
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====Comparison of series promoters: pVSW-3(GGG), pVSW-3(genome)====
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====Comparison of series promoters from pVSW-3(19) to pVSW-3(16)====
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In order to find a promoter that can function efficiently in Escherichia coli, we constructed <partinfo>BBa_K4907109</partinfo>_pSB3K3(pVSW-3(18)), <partinfo>BBa_K4907112</partinfo>_pSB3K3(pVSW-3(GGG)) and <partinfo>BBa_K4907122</partinfo>_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 <partinfo>BBa_K4907109</partinfo>_pSB3K3 showing the highest efficiency.
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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><imgsrc="hhttps://static.igem.wiki/teams/4907/wiki/parts/jincheng/biaozhen/ggg-gemone.png" width="400px"></html></center>
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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 promoters pVSW-3(18), pVSW-3(GGG) and pVSW-3(genome)</B></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>
 +
 
 +
====The optimal temperature of VSW-3 system====
 +
Since the low-temperature inducible expression, or a cold-responsive expression pattern, was the need for achieving the goal of anti-icing, the optimal temperature of the VSW-3 system should be investigated as well. The bacteria (BL21(DE3)) harboring VSW-3 RNAP and the reporting circuits of pVSW-3(18) was cultivated at different temperatures after induction respectively. Among the temperatures we tested, the VSW-3 system has the strongest activity at 25 °C (at least 4-fold higher than other groups) (Fig. 4), which was consistent with the results <i>in vitro</i> (3). Besides, the VSW-3 RNAP functioned better at 15 °C than 30 °C and physiological 37 °C, which implied the obvious <b>low-temperature preference</b> of this RNA polymerase. Due to the limited time and the number of available thermostats in our lab, more detailed temperature gradients need to be examined and we hoped that this could be achieved in the future.
 +
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/min.png" width="300px"></html></center>
 +
<center><b>Fig. 4 Characterizations of the temperature effect for VSW-3 system (a) and T7 system (b).</b></center>
  
 
===Reference===
 
===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).
+
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 <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).
  
−
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).
+
3.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).
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 12:44, 12 October 2023


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 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(18) 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(18) 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-1199 bp (lane K4907108).

Fig. 2 The result of colony PCR. Plasmid BBa_K4907108_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).

The optimal temperature of VSW-3 system

Since the low-temperature inducible expression, or a cold-responsive expression pattern, was the need for achieving the goal of anti-icing, the optimal temperature of the VSW-3 system should be investigated as well. The bacteria (BL21(DE3)) harboring VSW-3 RNAP and the reporting circuits of pVSW-3(18) was cultivated at different temperatures after induction respectively. Among the temperatures we tested, the VSW-3 system has the strongest activity at 25 °C (at least 4-fold higher than other groups) (Fig. 4), which was consistent with the results in vitro (3). Besides, the VSW-3 RNAP functioned better at 15 °C than 30 °C and physiological 37 °C, which implied the obvious low-temperature preference of this RNA polymerase. Due to the limited time and the number of available thermostats in our lab, more detailed temperature gradients need to be examined and we hoped that this could be achieved in the future.

Fig. 4 Characterizations of the temperature effect for VSW-3 system (a) and T7 system (b).

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).

3.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]