Difference between revisions of "Part:BBa K4907115"

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Based on the split-intein (3) combined with the novel VSW-3 system. In our design, the VSW-3 RNAP was split into two halves and fused to the split intein SspC and NpuN respectively.  
 
Based on the split-intein (3) combined with the novel VSW-3 system. In our design, the VSW-3 RNAP was split into two halves and fused to the split intein SspC and NpuN respectively.  
 
===Usage and design===
 
===Usage and design===
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We then turned to investigate the function of split intein and if the junction would result in an intact form of the polymerase with regaining normal function. The characterization circuit (<partinfo>BBa_K4907117</partinfo>) was constructed on the backbone pSB1C3 by placing the SspC-VSW-3 RNAPC and VSW-3 RNAPN-NpuN under the control of <i>L</i>-arabinose induced promoter BBa_I0500 in a bicistronic pattern.
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We built BBa K4907115_pSB1C3 and BBa K4907116_pSB1C3 to show that half of the polymerase alone can't function.
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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>O
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/i0500-b0034-vsw-3-rnapn-npun-b0015.png" width="400px"></html></center>
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<center><html><B>Fig. 1 Gene circuit of BBa K4907115 </B></html></center>
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Each fusion half was placed under the control of <i>cspA</i> promoter (pCspA). In this time, the two pCspA promoters acted as inputs while the pVSW-3(18) promoter played the role of output with the target genes placed downstream. Theoretically, leakage expression will occur at a certain probability for a single pCspA as output, however, <b>when the pVSW-3(18) is set as the output, the leakage at high temperatures will rarely happen due to the low-temperature preference of VSW-3 RNAP</b> even if the leakage of two pCspA promoters occur.
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<center><b>Fig. 9 Structure prediction. a</b> The predicted structure of VSW-3 RNAP. The split site was colored in blue. <b>b</b> The predicted structure of VSW-3 RNAPN-NpuN (<partinfo>BBa_K4907018</partinfo>). The NpuN was colored light pink. <b>c</b> The predicted structure of SspC-VSW-3 RNAPC (<partinfo>BBa_K4907017</partinfo>). The SspC was colored light pink.</center>
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===Characterization===
 
===Characterization===
 
====Agarose gel electrophoresis (AGE)====
 
====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-1201 bp (lane K4907130).
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When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-3427 bp (lane K4907115).
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/bba-k4907130-p.png" width="400px"></html></center>
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/115.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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<center><html><B>Fig. 2 The result of colony PCR. Plasmid BBa_K4907115_pSB1C3 </B></html></center>
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====The induction effect of spilt polymerase====
 
====The induction effect of spilt polymerase====
 
For careful verification, we preliminarily tested whether the split form of this VSW-3 RNAP could activate the pVSW-3(18) promoter or not. Each split half was placed under the control of <i>L</i>-arabinose induced promoter BBa_I0500 then constructed the expressing circuit, <partinfo>BBa_K4907115</partinfo> and <partinfo>BBa_K4907116</partinfo> on the backbone pSB1C3. The VSW-3 RNAP-expressing plasmid (<partinfo>BBa_K4907114</partinfo>_pSB1C3), and the split halves-expressing plasmids or the control (BBa_I0500) were co-transformed with the pVSW-3(18) reporting circuit (<partinfo>BBa_K4907108</partinfo>) into BL21(DE3), respectively. After induction at 25 °C for 12 h, both the group of VSW-3 RNAPC-NpuN and SspC-VSW-3 RNAPN showed no output signals like the control group, which were much lower than that of the intact VSW-3 RNAP (Fig. 10). Based on this observation, it was convinced that <b>the single half of the split RNA polymerase cannot function to trigger the expression of pVSW-3(18) promoter.</b>
 
For careful verification, we preliminarily tested whether the split form of this VSW-3 RNAP could activate the pVSW-3(18) promoter or not. Each split half was placed under the control of <i>L</i>-arabinose induced promoter BBa_I0500 then constructed the expressing circuit, <partinfo>BBa_K4907115</partinfo> and <partinfo>BBa_K4907116</partinfo> on the backbone pSB1C3. The VSW-3 RNAP-expressing plasmid (<partinfo>BBa_K4907114</partinfo>_pSB1C3), and the split halves-expressing plasmids or the control (BBa_I0500) were co-transformed with the pVSW-3(18) reporting circuit (<partinfo>BBa_K4907108</partinfo>) into BL21(DE3), respectively. After induction at 25 °C for 12 h, both the group of VSW-3 RNAPC-NpuN and SspC-VSW-3 RNAPN showed no output signals like the control group, which were much lower than that of the intact VSW-3 RNAP (Fig. 10). Based on this observation, it was convinced that <b>the single half of the split RNA polymerase cannot function to trigger the expression of pVSW-3(18) promoter.</b>
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/vsw-3-rnap/fig10.png" width="300px"></html></center>
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<center><b>Fig. 10 Characterizations for testing the activity of different forms of VSW-3 RNAP at 25 °C in BL21(DE3).</b> <i>p</i>-value: no significance (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****).</center>
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<center><b>Fig. 3 Characterizations for testing the activity of different forms of VSW-3 RNAP at 25 °C in BL21(DE3).</b> <i>p</i>-value: no significance (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****).</center>
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===Reference===
 
===Reference===

Latest revision as of 12:15, 12 October 2023


I0500-B0034-vsw-3 rnapN-npuN-B0015

Biology

VSW-3 RNAP

The VSW-3 RNAP is a novel single-subunit RNA polymerase encoded by the chillophilic phage VSW-3, which was first characterized in vitro in 2022. VSW-3 RNAP showed a good low-temperature performance, producing fewer terminal and full-length dsRNA byproducts than the T7 RNAP transcript in vitro (1). Moreover, the in vitro transcription products of VSW-3 RNAP were used to prepare mRNA for mRNA therapy in vivo due to the superior protein expression levels of VSW-3 RNA transcripts, compared to T7 RNAP transcripts (2).

VSW-3 RNAPN-NpuN and SspC VSW-3 RNAPC

Based on the split-intein (3) combined with the novel VSW-3 system. In our design, the VSW-3 RNAP was split into two halves and fused to the split intein SspC and NpuN respectively.

Usage and design

We built BBa K4907115_pSB1C3 and BBa K4907116_pSB1C3 to show that half of the polymerase alone can't function.

Fig. 1 Gene circuit of BBa K4907115

Characterization

Agarose gel electrophoresis (AGE)

When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-3427 bp (lane K4907115).

Fig. 2 The result of colony PCR. Plasmid BBa_K4907115_pSB1C3

The induction effect of spilt polymerase

For careful verification, we preliminarily tested whether the split form of this VSW-3 RNAP could activate the pVSW-3(18) promoter or not. Each split half was placed under the control of L-arabinose induced promoter BBa_I0500 then constructed the expressing circuit, BBa_K4907115 and BBa_K4907116 on the backbone pSB1C3. The VSW-3 RNAP-expressing plasmid (BBa_K4907114_pSB1C3), and the split halves-expressing plasmids or the control (BBa_I0500) were co-transformed with the pVSW-3(18) reporting circuit (BBa_K4907108) into BL21(DE3), respectively. After induction at 25 °C for 12 h, both the group of VSW-3 RNAPC-NpuN and SspC-VSW-3 RNAPN showed no output signals like the control group, which were much lower than that of the intact VSW-3 RNAP (Fig. 10). Based on this observation, it was convinced that the single half of the split RNA polymerase cannot function to trigger the expression of pVSW-3(18) promoter.

Fig. 3 Characterizations for testing the activity of different forms of VSW-3 RNAP at 25 °C in BL21(DE3). p-value: no significance (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****).

Reference

1. 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).

2.G. Wang et al., mRNA produced by VSW-3 RNAP has high-level translation efficiency with low inflammatory stimulation. Cell Insight 1, 100056 (2022).

3.L. Saleh, F. B. Perler, Protein splicing in cis and in trans. Chem Rec 6, 183-193 (2006).

4.G. Qing et al., Cold-shock induced high-yield protein production in Escherichia coli. Nature Biotechnology 22, 877-882 (2004).

5.B. Wang, R. I. Kitney, N. Joly, M. Buck, Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nature Communications 2, 508 (2011).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2687
    Illegal BglII site found at 2924
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 1926
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1449
    Illegal BsaI.rc site found at 2680
    Illegal SapI site found at 961