Difference between revisions of "Part:BBa K5348017"

 
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pYC-pKC-pL-mcherry
 
  
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===Usage and Biology===
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===Functional Parameters===
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<partinfo>BBa_K5348017 parameters</partinfo>
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    <title>pYC-pKC-pL-RBS0-mCherry (BBa_K5348017)</title>
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    <h2>pYC-pKC-pL-RBS0-mCherry (BBa_K5348017)</h2>
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    <h3>Summary</h3>
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    <p>
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        To reduce the leaky expression of the light-on induced system (BBa_K3447133), we reduced the strength of the RBS, which is connected to the target genes, and tested its light-controlled regulatory function using mCherry as a model protein. The following test results of the original RBS show that the element can regulate mCherry expression, but there is leakage.
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    </p>
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    <h3>Construction Design</h3>
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    <p>
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        This composite part consists of the pL-RBS0-mCherry (BBa_K5348008) and pTrc99k-backbone (BBa_K3999002), which was constructed in E. coli DH5α strain. With the pL light-control system, regulation of mCherry expression in the dark and under blue light can be achieved.
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    </p>
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5348/bba-k5348017/figure-1.jpg" alt="Figure 1. Schematic diagram of pYC-pKC-pL-RBS0-mCherry">
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        <div class="caption">Figure 1. Schematic diagram of pYC-pKC-pL-RBS0-mCherry</div>
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    </div>
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    <h3>Engineering Principle</h3>
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    <p>
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        The pL light-control system consists of several basic parts. Under dark condition, histidine kinase (YF1) phosphorylates FixJ (response regulator of histidine kinase), which activates PFixK2 (the target gene for transcription upon FixJ activation), driving the expression of the cI gene (λ phage repressor), which represses the transcription of its cognate promoter, PR (the cognate promoter of cI), and downstream genes cannot be expressed. Under blue light, the cI gene cannot be expressed, PR can be transcribed normally, and downstream genes can be expressed [1].
 +
    </p>
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    <h3>Experimental Approach</h3>
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    <p>
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        The plasmid construction scheme is shown in Figure 2A. We synthesized the pL element at GenScript and divided it into two fragments, pL-1 and pL-2, for synthesis. We amplified pL-1, pL-2-RBS(0) and RBS(0)-mCherry fragments, and then ligated the pL-2-RBS(0) and RBS(0)-mCherry fragments by overlapping PCR to obtain the pL-2-RBS(0)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(0)-mCherry fragments, and the pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(0)-mCherry plasmid (Figure 2B).
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    </p>
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5348/bba-k5348017/figure-2.jpg" alt="Figure 2. Construction results of pYC-pKC-pL-RBS(0)-mCherry plasmid">
 +
        <div class="caption">Figure 2. Construction results of pYC-pKC-pL-RBS(0)-mCherry plasmid. (A) Construction Strategy. (B) Colony PCR and sequencing results.</div>
 +
    </div>
 +
 
 +
    <h3>Measurement: Light Control Test</h3>
 +
    <p>
 +
        Subsequently, we conducted light-control tests on the strain containing the pYC-pKC-pL-RBS(0)-mCherry plasmid. We cultured the strains under dark condition and blue light irradiation, respectively, sampling at intervals to measure the RFU (relative fluorescence units) of the bacterial suspension. As shown in Figure 3, the test results verified that the pL light-control element could regulate mCherry expression under dark and blue light conditions. However, it was observed that the pL light-control system exhibited leakage, with detectable increases in mCherry RFU after culturing for more than 8 hours under dark conditions.
 +
    </p>
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5348/bba-k5348017/figure-3.jpg" alt="Figure 3. Light-control test results">
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        <div class="caption">Figure 3. Light-control test results.</div>
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    </div>
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    <h3>References</h3>
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    <p>[1] H, Mays RL, Hoffman SM, Avalos JL. Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS Synth Biol. 2021 Aug 20;10(8):2015-2029.</p>
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</body>
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</html>

Revision as of 11:02, 30 September 2024

pYC-pKC-pL-RBS0-mCherry



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 5855
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4560
    Illegal NgoMIV site found at 4632
    Illegal NgoMIV site found at 4722
    Illegal NgoMIV site found at 4740
    Illegal NgoMIV site found at 5232
    Illegal NgoMIV site found at 5525
    Illegal NgoMIV site found at 5619
    Illegal AgeI site found at 4274
    Illegal AgeI site found at 5400
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5289
    Illegal BsaI.rc site found at 4173
    Illegal SapI site found at 1
    Illegal SapI.rc site found at 3967


<!DOCTYPE html> pYC-pKC-pL-RBS0-mCherry (BBa_K5348017)

pYC-pKC-pL-RBS0-mCherry (BBa_K5348017)

Summary

To reduce the leaky expression of the light-on induced system (BBa_K3447133), we reduced the strength of the RBS, which is connected to the target genes, and tested its light-controlled regulatory function using mCherry as a model protein. The following test results of the original RBS show that the element can regulate mCherry expression, but there is leakage.

Construction Design

This composite part consists of the pL-RBS0-mCherry (BBa_K5348008) and pTrc99k-backbone (BBa_K3999002), which was constructed in E. coli DH5α strain. With the pL light-control system, regulation of mCherry expression in the dark and under blue light can be achieved.

Figure 1. Schematic diagram of pYC-pKC-pL-RBS0-mCherry
Figure 1. Schematic diagram of pYC-pKC-pL-RBS0-mCherry

Engineering Principle

The pL light-control system consists of several basic parts. Under dark condition, histidine kinase (YF1) phosphorylates FixJ (response regulator of histidine kinase), which activates PFixK2 (the target gene for transcription upon FixJ activation), driving the expression of the cI gene (λ phage repressor), which represses the transcription of its cognate promoter, PR (the cognate promoter of cI), and downstream genes cannot be expressed. Under blue light, the cI gene cannot be expressed, PR can be transcribed normally, and downstream genes can be expressed [1].

Experimental Approach

The plasmid construction scheme is shown in Figure 2A. We synthesized the pL element at GenScript and divided it into two fragments, pL-1 and pL-2, for synthesis. We amplified pL-1, pL-2-RBS(0) and RBS(0)-mCherry fragments, and then ligated the pL-2-RBS(0) and RBS(0)-mCherry fragments by overlapping PCR to obtain the pL-2-RBS(0)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(0)-mCherry fragments, and the pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(0)-mCherry plasmid (Figure 2B).

Figure 2. Construction results of pYC-pKC-pL-RBS(0)-mCherry plasmid
Figure 2. Construction results of pYC-pKC-pL-RBS(0)-mCherry plasmid. (A) Construction Strategy. (B) Colony PCR and sequencing results.

Measurement: Light Control Test

Subsequently, we conducted light-control tests on the strain containing the pYC-pKC-pL-RBS(0)-mCherry plasmid. We cultured the strains under dark condition and blue light irradiation, respectively, sampling at intervals to measure the RFU (relative fluorescence units) of the bacterial suspension. As shown in Figure 3, the test results verified that the pL light-control element could regulate mCherry expression under dark and blue light conditions. However, it was observed that the pL light-control system exhibited leakage, with detectable increases in mCherry RFU after culturing for more than 8 hours under dark conditions.

Figure 3. Light-control test results
Figure 3. Light-control test results.

References

[1] H, Mays RL, Hoffman SM, Avalos JL. Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS Synth Biol. 2021 Aug 20;10(8):2015-2029.