Difference between revisions of "Part:BBa K3823010"

 
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<partinfo>BBa_K3823010 short</partinfo>
 
<partinfo>BBa_K3823010 short</partinfo>
  
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===Purposes and Design===
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In our project, we wanted to construct a three-gear kill switch based on hydrogen sulfide sensing, so we needed a component that would allow both MazE and Dre recombinases to be regulated by an H<sub>2</sub>S-mediated switch.  In this element, MazE and Dre are controlled by Pcstr ([https://parts.igem.org/Part:BBa_K3823008 BBa_K3823008]), which is repressed by CstR([https://parts.igem.org/Part:BBa_K3823006 BBa_K3823006]).CstR is an HS<sub>n</sub>H sensitive regulator.
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Its position in our plasmid construction is shown here(Figure 1.).
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[[Image:BBa K38230012-5.png|center|thumb|600px|Figure 1.  The modified plasmid construction flow chart]]
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We contacted Professor Huaiwei Liu from Shandong University based on literature and fortunately got his help. Using the plasmid he donated as a template, we successfully amplified the CstR-Pcstr sequence(Figure 2A) and used it for our plasmid construction.
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We successfully constructed the plasmid CstR-Pcstr-MazE -Dre and amplified CstR-Pcstr-MazE-Dre([https://parts.igem.org/Part:BBa_K3823010 BBa_K3823010])(Figure 2B).Then we cloned CstR-Pcstr-MazE-Dre to pACYC-rox-ter-rox-MazF.
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[[Image:BBa K3823006-4.png|center|thumb|600px|Figure 2. Agarose electrophoresis]]
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However, according to the sequencing results, we found that Pcstr was missing(not in the PCR product), probably due to the complexity of each block. Meanwhile, we found the terminator between the two rox sites was partly moved after we cultured the final plasmid. We assumed that it could be the leakage of Pcstr that turned on the expression of Dre, which then cut off the terminator. This means we need to optimize our design.
  
 
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Revision as of 03:50, 21 October 2021


MazE antitoxin and Dre recombinase regulated by a hydrogen sulfide switch

Purposes and Design

In our project, we wanted to construct a three-gear kill switch based on hydrogen sulfide sensing, so we needed a component that would allow both MazE and Dre recombinases to be regulated by an H2S-mediated switch. In this element, MazE and Dre are controlled by Pcstr (BBa_K3823008), which is repressed by CstR(BBa_K3823006).CstR is an HSnH sensitive regulator.

Its position in our plasmid construction is shown here(Figure 1.).
Figure 1. The modified plasmid construction flow chart

We contacted Professor Huaiwei Liu from Shandong University based on literature and fortunately got his help. Using the plasmid he donated as a template, we successfully amplified the CstR-Pcstr sequence(Figure 2A) and used it for our plasmid construction. We successfully constructed the plasmid CstR-Pcstr-MazE -Dre and amplified CstR-Pcstr-MazE-Dre(BBa_K3823010)(Figure 2B).Then we cloned CstR-Pcstr-MazE-Dre to pACYC-rox-ter-rox-MazF.

Figure 2. Agarose electrophoresis

However, according to the sequencing results, we found that Pcstr was missing(not in the PCR product), probably due to the complexity of each block. Meanwhile, we found the terminator between the two rox sites was partly moved after we cultured the final plasmid. We assumed that it could be the leakage of Pcstr that turned on the expression of Dre, which then cut off the terminator. This means we need to optimize our design.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 646
    Illegal PstI site found at 265
    Illegal PstI site found at 953
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 646
    Illegal PstI site found at 265
    Illegal PstI site found at 953
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 646
    Illegal BglII site found at 242
    Illegal BglII site found at 1828
    Illegal BamHI site found at 1006
    Illegal BamHI site found at 2342
    Illegal XhoI site found at 2300
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 646
    Illegal PstI site found at 265
    Illegal PstI site found at 953
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 646
    Illegal PstI site found at 265
    Illegal PstI site found at 953
  • 1000
    COMPATIBLE WITH RFC[1000]