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    <h2>BBa_K5348004 (pL-RBS1)</h2>
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    <h3>Summary</h3>
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    <p>
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        To reduce the leaky expression of this part, we reduced the strength of the RBS  by 9-fold, which is connected to the target genes. We tested the fluorescent protein mCherry as model protein.
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    <h3>Construction Design</h3>
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===Functional Parameters===
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        To reduce the leaky expression of the light-inducible induction system (BBa_K3447133, hereafter referred to as the pL), we reduced the intensity of the RBS linked to the target gene in this element by 9-fold (RBS1). The composition of this element is shown below.
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        <img src="https://static.igem.wiki/teams/5348/bba-k5348004/figure-1.jpg" alt="Figure 1. Schematic diagram of the pL-RBS1">
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        <div class="caption">Figure 1. Schematic diagram of the pL-RBS1</div>
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    <h3>Engineering Principle</h3>
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        Under dark condition, histidine kinase (YF1) phosphorylates FixJ (response regulator of histidine kinase), which activates P<sub>FixK2</sub> (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, P<sub>R</sub> (the cognate promoter of cI), and downstream genes cannot be expressed. Under blue light, the cI gene cannot be expressed, P<sub>R</sub> can be transcribed normally, and downstream genes can be expressed [1].
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    <h3>Experimental Approach</h3>
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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(1) and RBS(1)-mCherry fragments, and then ligated the pL-2-RBS(1) and RBS(1)-mCherry fragments by overlapping PCR to obtain pL-2-RBS(1)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(1)-mCherry fragments, and pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(1)-mCherry plasmid (Figure 2B).
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        <img src="https://static.igem.wiki/teams/5348/bba-k5348009/figure-2.jpg" alt="Figure 2.Construction results of pYC-pKC-pL-RBS(1)-mCherry plasmid.">
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        <div class="caption">Figure 2.Construction results of pYC-pKC-pL-RBS(1)-mCherry plasmid</div>
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    <h3>Measurement: Light Control Test</h3>
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    <p>
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        Subsequently, we conducted light-control tests on the strain containing pYC-pKC-pL-RBS(1)-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. As the RBS strength decreased, the RFU of mCherry decreased accordingly, indicating that the RBS replacement strategy can achieve regulation of the pL light-control system.
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5348/bba-k5348009/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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    <h3>Reference</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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Latest revision as of 13:03, 2 October 2024


pL-RBS1


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 1874
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 587
    Illegal NgoMIV site found at 659
    Illegal NgoMIV site found at 749
    Illegal NgoMIV site found at 767
    Illegal NgoMIV site found at 1259
    Illegal NgoMIV site found at 1552
    Illegal NgoMIV site found at 1646
    Illegal AgeI site found at 301
    Illegal AgeI site found at 1427
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1316
    Illegal BsaI.rc site found at 200

<!DOCTYPE html> BBa_K5348004 (pL-RBS1)

BBa_K5348004 (pL-RBS1)

Summary

To reduce the leaky expression of this part, we reduced the strength of the RBS by 9-fold, which is connected to the target genes. We tested the fluorescent protein mCherry as model protein.

Construction Design

To reduce the leaky expression of the light-inducible induction system (BBa_K3447133, hereafter referred to as the pL), we reduced the intensity of the RBS linked to the target gene in this element by 9-fold (RBS1). The composition of this element is shown below.

Figure 1. Schematic diagram of the pL-RBS1
Figure 1. Schematic diagram of the pL-RBS1

Engineering Principle

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(1) and RBS(1)-mCherry fragments, and then ligated the pL-2-RBS(1) and RBS(1)-mCherry fragments by overlapping PCR to obtain pL-2-RBS(1)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(1)-mCherry fragments, and pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(1)-mCherry plasmid (Figure 2B).

Figure 2.Construction results of pYC-pKC-pL-RBS(1)-mCherry plasmid.
Figure 2.Construction results of pYC-pKC-pL-RBS(1)-mCherry plasmid

Measurement: Light Control Test

Subsequently, we conducted light-control tests on the strain containing pYC-pKC-pL-RBS(1)-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. As the RBS strength decreased, the RFU of mCherry decreased accordingly, indicating that the RBS replacement strategy can achieve regulation of the pL light-control system.

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

Reference

[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.