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     <h3>Summary</h3>
 
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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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         We have added new experimental data to the already existing part: <b>BBa_K3447133 (light-on induced system)</b>. We used mCherry as a reporter gene and therefore added a new composite part: <b>BBa_K5348008 </b>, to test the light control system.
 
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     <h3>Construction Design</h3>
 
     <h3>Construction Design</h3>
 
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         This composite part consists of the BBa_K3447133 (hereafter referred to as the pL) and fluorescent protein mCherry (BBa_K3822002). With the pL light-control system, regulation of mCherry expression in the dark and under blue light can be achieved.
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         This composite part consists of the BBa_K3447133 (hereafter referred to as the pL-RBS0) and fluorescent protein mCherry (BBa_K3822002). With the pL light-control system, regulation of mCherry expression in the dark and under blue light can be achieved.
 
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Latest revision as of 13:16, 1 October 2024


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 1882
  • 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


BBa_K5348008 (pL-RBS0-mCherry)

BBa_K5348008 (pL-RBS0-mCherry)

Summary

We have added new experimental data to the already existing part: BBa_K3447133 (light-on induced system). We used mCherry as a reporter gene and therefore added a new composite part: BBa_K5348008 , to test the light control system.

Construction Design

This composite part consists of the BBa_K3447133 (hereafter referred to as the pL-RBS0) and fluorescent protein mCherry (BBa_K3822002). With the pL light-control system, regulation of mCherry expression in the dark and under blue light can be achieved.

Engineering Principle

The pL light-control system consists of several basic parts. Under dark conditions, 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].

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

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 pL-2-RBS(0)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(0)-mCherry fragments, and 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 pYC-pKC-pL-RBS(0)-mCherry plasmid. We cultured the strains under dark conditions 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.