Difference between revisions of "Part:BBa K5348034"

 
 
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pLEVI2.0-MazF
 
  
  
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===Usage and Biology===
 
  
 
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===Functional Parameters===
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<partinfo>BBa_K5348034 parameters</partinfo>
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    <title>pLEVI2.0-MazF (BBa_K5348034)</title>
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    <h2>Construction Design</h2>
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        This composite part consists of pLEVI2.0 (BBa_K5348032) and toxin protein MazF (BBa_K1096002), which was constructed in <i>E. coli</i> DH5α strain.
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    <h2>Engineering Principle</h2>
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    <p>
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        Under blue light irradiation, the LEVI domain undergoes a conformational change, forming a protein dimer. This dimer binds to its homologous operator sequence, inhibiting PColE promoter activity. Consequently, the cI repressor cannot be expressed, allowing the target protein to be expressed. In dark conditions, the PColE promoter initiates transcription and expression of the cI repressor, thereby inhibiting target gene transcription [1]. Additionally, we used the CloDF13 ori, a low-copy replication origin more suitable for constructing plasmids containing toxic genes. Our modified light-control system, LEVI2.0, is shown in Figure 1.
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5348/bba-k5348034/figure-1.jpg" alt="Figure 1. Schematic diagram of pLEVI2.0-MazF">
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        <div class="caption">Figure 1. Schematic diagram of pLEVI2.0-MazF.</div>
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    <h2>Experimental Approach</h2>
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        Using pYC-pKC-pL-MazF plasmid as a template, we introduced AatII and XhoI restriction sites upstream and downstream of the MazF fragment, obtaining the AatII-MazF-XhoI fragment. We then performed double digestion (AatII and XhoI) on both the AatII-MazF-XhoI fragment and pLEVI2.0 plasmid to obtain the digested fragment and backbone (Figure 2).
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        <img src="https://static.igem.wiki/teams/5348/bba-k5348034/figure-2.jpg" alt="Figure 2. Construction results of the pLEVI2.0-MazF plasmid">
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        <div class="caption">Figure 2. Construction results of the pLEVI2.0-MazF plasmid. (A) Design of pLEVI2.0-MazF plasmid construction. (B) Amplification results of the MazF fragment. (C) Enzyme digestion of the vector.</div>
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        Due to time constraints, we have not been able to complete the linkage of the fragments to the backbone, transformation, and light-control tests. Our expected result is that the strain does not express or expresses a small amount of MazF when cultured under dark conditions. In contrast, under high-intensity blue light, the strain can express MazF protein, resulting in impaired strain growth.
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    <h2>References</h2>
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    <p>[1] Chen, X., Liu, R., Ma, Z. et al. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. <i>Cell Res.</i> 2016, 26, 854–857.</p>
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Latest revision as of 14:34, 30 September 2024


pLEVI2.0-MazF



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1332
    Illegal NheI site found at 3548
    Illegal NheI site found at 3571
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 71
    Illegal XhoI site found at 120
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 90
    Illegal NgoMIV site found at 585
    Illegal AgeI site found at 332
    Illegal AgeI site found at 4253
  • 1000
    COMPATIBLE WITH RFC[1000]


pLEVI2.0-MazF (BBa_K5348034)

Construction Design

This composite part consists of pLEVI2.0 (BBa_K5348032) and toxin protein MazF (BBa_K1096002), which was constructed in E. coli DH5α strain.

Engineering Principle

Under blue light irradiation, the LEVI domain undergoes a conformational change, forming a protein dimer. This dimer binds to its homologous operator sequence, inhibiting PColE promoter activity. Consequently, the cI repressor cannot be expressed, allowing the target protein to be expressed. In dark conditions, the PColE promoter initiates transcription and expression of the cI repressor, thereby inhibiting target gene transcription [1]. Additionally, we used the CloDF13 ori, a low-copy replication origin more suitable for constructing plasmids containing toxic genes. Our modified light-control system, LEVI2.0, is shown in Figure 1.

Figure 1. Schematic diagram of pLEVI2.0-MazF
Figure 1. Schematic diagram of pLEVI2.0-MazF.

Experimental Approach

Using pYC-pKC-pL-MazF plasmid as a template, we introduced AatII and XhoI restriction sites upstream and downstream of the MazF fragment, obtaining the AatII-MazF-XhoI fragment. We then performed double digestion (AatII and XhoI) on both the AatII-MazF-XhoI fragment and pLEVI2.0 plasmid to obtain the digested fragment and backbone (Figure 2).

Figure 2. Construction results of the pLEVI2.0-MazF plasmid
Figure 2. Construction results of the pLEVI2.0-MazF plasmid. (A) Design of pLEVI2.0-MazF plasmid construction. (B) Amplification results of the MazF fragment. (C) Enzyme digestion of the vector.

Due to time constraints, we have not been able to complete the linkage of the fragments to the backbone, transformation, and light-control tests. Our expected result is that the strain does not express or expresses a small amount of MazF when cultured under dark conditions. In contrast, under high-intensity blue light, the strain can express MazF protein, resulting in impaired strain growth.

References

[1] Chen, X., Liu, R., Ma, Z. et al. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Res. 2016, 26, 854–857.