Difference between revisions of "Part:BBa K5068007"

 
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p15A-op-merR-bspA-pcpS
 
  
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===Usage and Biology===
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===Functional Parameters===
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<partinfo>BBa_K5068007 parameters</partinfo>
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    <meta charset="UTF-8">
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    <title>BBa_K5068007 (p15A-op-merR-bspA-pcpS)</title>
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    <style>
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            max-width: 80%; /* Adjust this percentage to change size relative to text */
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<body>
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    <h2>New Improved Part: BBa_K5068007 (p15A-op-merR-bspA-pcpS)</h2>
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    <h3>Summary</h3>
 +
    <p>
 +
        The <em>bapA</em> and <em>pcpS</em> genes play important roles in the production and transport of pigments. The <em>bapA</em> gene encodes a protein involved in the synthesis of the blue pigment indigo (McNerney, 2017), while the <em>pcpS</em> gene encodes a PPTase that activates acyl carrier proteins, facilitating the effective transport of the synthesized pigment within the cell. These two genes typically work in synergy, with <em>bapA</em> synthesizing indigo and <em>pcpS</em> enhancing the transport and stability of the pigment, thereby increasing the bacteria's survival in specific environments (Xie, 2019). In a heterologous expression system, the combination of <em>bspA</em> (a single-module non-ribosomal peptide synthetase) and <em>pcpS</em> enables the successful biosynthesis of indigo in <em>Escherichia coli</em>, demonstrating their collaborative role in the pigment biosynthetic pathway.
 +
        Based on BBa_K1420004 (merR), we constructed a new combination plasmid BBa_K5068007 (p15A-op-merR-bspA-pcpS). This plasmid can be used to detect Hg. In addition, we also performed sequence optimization to improve the sensitivity of mercury detection.
 +
    </p>
 +
 
 +
    <h3>Construction Design</h3>
 +
    <p>
 +
        The p15A-op-merR-bspA-pcpS is composed of BBa_K5068003 (op-merR), BBa_M36245 (Pcad), BBa_K4605002 (bspA), BBa_K5068002 (pcpS), and BBa_K5067004 (p15A). We connected the gene to the vector through homologous recombination (Fig. 1), and then transferred it into <em>E. coli</em> DH5α for copying.
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    </p>
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 +
    <!-- Figure 1 -->
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5068/bba-k5068007/1.png" alt="Figure 1: The map of p15A-op-merR-bspA-pcpS">
 +
        <div class="caption">Fig. 1. The map of p15A-op-merR-bspA-pcpS</div>
 +
    </div>
 +
 
 +
    <h3>Engineering Principle</h3>
 +
    <p>
 +
        The plasmid p15A-op-merR-bspA-pcpS corresponds to transcriptional activation mediated by Hg (II), enabling the expression of indigo pigment in the presence of Hg (II). Upon transforming this plasmid into competent cells, a biosensor for Hg (II) detection can be established (Fig. 2). This biosensor utilizes the production of indigo pigment as a visual indicator, allowing for the straightforward identification of mercury contamination in various samples.
 +
    </p>
 +
 
 +
    <!-- Figure 2 -->
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5068/bba-k5068007/2.png" alt="Figure 2: The principle of mercury detection">
 +
        <div class="caption">Fig. 2. The principle of mercury detection</div>
 +
    </div>
 +
 
 +
    <h3>Experimental Approach</h3>
 +
    <p>
 +
        The plasmid backbone p15A (BBa_K5068009) was linearized using inverse PCR. Subsequently, the genes op-merR-merTPCAD and bspA-pcpS were amplified via PCR, yielding three fragments necessary for vector construction (Fig. 3). Each fragment had homologous arms introduced at both ends through the primers, which can be seen in Fig. 3.
 +
    </p>
 +
 
 +
    <!-- Figure 3 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5068/bba-k5068007/3.png" alt="Figure 3: Electropherogram of fragments required for vector construction.">
 +
        <div class="caption">Fig. 3. Electropherogram of fragments required for vector construction. The length of p15A is 5061bp, the length of merR is 568bp, and the lengths of bspA and pcpS are 4643bp.</div>
 +
    </div>
 +
 
 +
    <p>
 +
        These three fragments were then joined using homologous recombination. Because each pair of adjacent fragments contained homologous sequences, a circular structure was ultimately formed, resulting in the recombinant plasmid p15A-op-merR-bspA-pcpS (Fig. 4A).
 +
        The constructed recombinant plasmid was subsequently transformed into competent <em>E. coli</em> strains TOP10 and BL21 (Fig. 4B), and the transformants were verified. Results from monoclonal PCR validation (Fig. 4C) and sequencing alignment (Fig. 4D) demonstrated that the sequences were highly consistent with the target sequence, with no significant mutations or insertions/deletions observed, confirming the successful transformation of the plasmid into <em>E. coli</em>. Ultimately, the recombinant plasmid was obtained, along with positive clones containing the recombinant plasmid in both the TOP10 and BL21 strains.
 +
    </p>
 +
 
 +
    <!-- Figure 4 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5068/bba-k5068007/4.png" alt="Figure 4: Transformation process and transformant validation">
 +
        <div class="caption">Fig. 4. Transformation process and transformant validation</div>
 +
    </div>
 +
 
 +
    <h3>Characterization</h3>
 +
    <p>
 +
        We transformed the plasmid into <em>E. coli</em> BL21 and coated it onto Hg<sup>2+</sup> plates of different concentrations, without Hg<sup>2+</sup> as the control group. In Figure 5, we can see blue colonies on the plates with 10 μmol Hg<sup>2+</sup> and 20 μmol Hg<sup>2+</sup>, but the plates without mercury ions did not turn blue. This indicates that our mercury detection sensor is working, while also detecting mercury ions.
 +
    </p>
 +
 
 +
    <!-- Figure 5 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5068/bba-k5068007/5.png" alt="Figure 5: The effectiveness of mercury detection at different concentrations of Hg2+">
 +
        <div class="caption">Fig. 5. The effectiveness of mercury detection at different concentrations of Hg<sup>2+</sup></div>
 +
    </div>
 +
 
 +
    <p>
 +
        Then the blue colonies were inoculated into the culture medium of different concentrations of Hg<sup>2+</sup>, and the absorbance A<sub>600</sub> was measured after centrifugation. According to Fig. 6A, the color gradually became blue with the increase of Hg<sup>2+</sup> concentration, and Fig. 6B showed that the A<sub>600</sub> of 5, 10, 20 μmol Hg<sup>2+</sup> was significantly higher than that of 0 μmol Hg<sup>2+</sup>, indicating that the indigo gene bspA-pcpS was expressed and the mercury detection system was sensitive.
 +
    </p>
 +
 
 +
    <!-- Figure 6 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5068/bba-k5068007/6.png" alt="Figure 6: The effectiveness of mercury detection at different concentrations of Hg2+">
 +
        <div class="caption">Fig. 6. The effectiveness of mercury detection at different concentrations of Hg<sup>2+</sup></div>
 +
    </div>
 +
 
 +
    <h3>References</h3>
 +
    <p>
 +
        1. McNerney MP, Michel CL, Kishore K, Standeven J, Styczynski MP (2019) Dynamic and tunable metabolite control for robust minimal-equipment assessment of serum zinc. <em>Nat Commun</em> 10(1):5514. <a href="https://doi.org/10.1038/s41467-019-13454-1">https://doi.org/10.1038/s41467-019-13454-1</a><br>
 +
        2. Xie Z, Zhang Z, Cao Z, Chen M, Li P, Liu W, Qin H, Zhao X, Tao Y, Chen Y (2017) An external substrate-free blue/white screening system in <em>Escherichia coli</em>. <em>Appl Microbiol Biotechnol</em> 101(9):3811–3820.
 +
    </p>
 +
 
 +
</body>
 +
</html>

Revision as of 01:12, 29 September 2024


p15A-op-merR-bspA-pcpS



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 5125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1337
    Illegal BamHI site found at 3589
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3615
    Illegal NgoMIV site found at 11153
    Illegal NgoMIV site found at 11291
    Illegal AgeI site found at 1172
    Illegal AgeI site found at 2829
    Illegal AgeI site found at 3938
    Illegal AgeI site found at 5211
    Illegal AgeI site found at 5535
  • 1000
    COMPATIBLE WITH RFC[1000]


<!DOCTYPE html> BBa_K5068007 (p15A-op-merR-bspA-pcpS)

New Improved Part: BBa_K5068007 (p15A-op-merR-bspA-pcpS)

Summary

The bapA and pcpS genes play important roles in the production and transport of pigments. The bapA gene encodes a protein involved in the synthesis of the blue pigment indigo (McNerney, 2017), while the pcpS gene encodes a PPTase that activates acyl carrier proteins, facilitating the effective transport of the synthesized pigment within the cell. These two genes typically work in synergy, with bapA synthesizing indigo and pcpS enhancing the transport and stability of the pigment, thereby increasing the bacteria's survival in specific environments (Xie, 2019). In a heterologous expression system, the combination of bspA (a single-module non-ribosomal peptide synthetase) and pcpS enables the successful biosynthesis of indigo in Escherichia coli, demonstrating their collaborative role in the pigment biosynthetic pathway. Based on BBa_K1420004 (merR), we constructed a new combination plasmid BBa_K5068007 (p15A-op-merR-bspA-pcpS). This plasmid can be used to detect Hg. In addition, we also performed sequence optimization to improve the sensitivity of mercury detection.

Construction Design

The p15A-op-merR-bspA-pcpS is composed of BBa_K5068003 (op-merR), BBa_M36245 (Pcad), BBa_K4605002 (bspA), BBa_K5068002 (pcpS), and BBa_K5067004 (p15A). We connected the gene to the vector through homologous recombination (Fig. 1), and then transferred it into E. coli DH5α for copying.

Figure 1: The map of p15A-op-merR-bspA-pcpS
Fig. 1. The map of p15A-op-merR-bspA-pcpS

Engineering Principle

The plasmid p15A-op-merR-bspA-pcpS corresponds to transcriptional activation mediated by Hg (II), enabling the expression of indigo pigment in the presence of Hg (II). Upon transforming this plasmid into competent cells, a biosensor for Hg (II) detection can be established (Fig. 2). This biosensor utilizes the production of indigo pigment as a visual indicator, allowing for the straightforward identification of mercury contamination in various samples.

Figure 2: The principle of mercury detection
Fig. 2. The principle of mercury detection

Experimental Approach

The plasmid backbone p15A (BBa_K5068009) was linearized using inverse PCR. Subsequently, the genes op-merR-merTPCAD and bspA-pcpS were amplified via PCR, yielding three fragments necessary for vector construction (Fig. 3). Each fragment had homologous arms introduced at both ends through the primers, which can be seen in Fig. 3.

Figure 3: Electropherogram of fragments required for vector construction.
Fig. 3. Electropherogram of fragments required for vector construction. The length of p15A is 5061bp, the length of merR is 568bp, and the lengths of bspA and pcpS are 4643bp.

These three fragments were then joined using homologous recombination. Because each pair of adjacent fragments contained homologous sequences, a circular structure was ultimately formed, resulting in the recombinant plasmid p15A-op-merR-bspA-pcpS (Fig. 4A). The constructed recombinant plasmid was subsequently transformed into competent E. coli strains TOP10 and BL21 (Fig. 4B), and the transformants were verified. Results from monoclonal PCR validation (Fig. 4C) and sequencing alignment (Fig. 4D) demonstrated that the sequences were highly consistent with the target sequence, with no significant mutations or insertions/deletions observed, confirming the successful transformation of the plasmid into E. coli. Ultimately, the recombinant plasmid was obtained, along with positive clones containing the recombinant plasmid in both the TOP10 and BL21 strains.

Figure 4: Transformation process and transformant validation
Fig. 4. Transformation process and transformant validation

Characterization

We transformed the plasmid into E. coli BL21 and coated it onto Hg2+ plates of different concentrations, without Hg2+ as the control group. In Figure 5, we can see blue colonies on the plates with 10 μmol Hg2+ and 20 μmol Hg2+, but the plates without mercury ions did not turn blue. This indicates that our mercury detection sensor is working, while also detecting mercury ions.

Figure 5: The effectiveness of mercury detection at different concentrations of Hg2+
Fig. 5. The effectiveness of mercury detection at different concentrations of Hg2+

Then the blue colonies were inoculated into the culture medium of different concentrations of Hg2+, and the absorbance A600 was measured after centrifugation. According to Fig. 6A, the color gradually became blue with the increase of Hg2+ concentration, and Fig. 6B showed that the A600 of 5, 10, 20 μmol Hg2+ was significantly higher than that of 0 μmol Hg2+, indicating that the indigo gene bspA-pcpS was expressed and the mercury detection system was sensitive.

Figure 6: The effectiveness of mercury detection at different concentrations of Hg2+
Fig. 6. The effectiveness of mercury detection at different concentrations of Hg2+

References

1. McNerney MP, Michel CL, Kishore K, Standeven J, Styczynski MP (2019) Dynamic and tunable metabolite control for robust minimal-equipment assessment of serum zinc. Nat Commun 10(1):5514. https://doi.org/10.1038/s41467-019-13454-1
2. Xie Z, Zhang Z, Cao Z, Chen M, Li P, Liu W, Qin H, Zhao X, Tao Y, Chen Y (2017) An external substrate-free blue/white screening system in Escherichia coli. Appl Microbiol Biotechnol 101(9):3811–3820.