Difference between revisions of "Part:BBa K4119008"
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<partinfo>BBa_K4119008 parameters</partinfo>
 
<partinfo>BBa_K4119008 parameters</partinfo>
 
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    <p style="font-size: 180%; font-weight: bold;">Short Description: lactose-inducing promoter+ CRISPR array</p>
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    <p>The upper and lower homology arms (HR-UP, HR-DOWN) were constructed at about 500 bp upstream and downstream of
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        the PerR sequence of the Clostridium tyrobutyricum genome, plus a lactose-inducing promoter and a spacer
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        sequence derived from the PerR , generating the CRISPR array .Then inserted it into the pMTL82151 plasmid, the
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        successful plasmid is transformed into E. coli CA434, and introduced into Clostridium tyrobutyricum, by lactose
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        induction to make the spacer hit the perR sequence on the target genome, thereby utilizing the endogenous
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        CRISPR-Cas system knocks the perR out , eliminates the impediment effect of perR on the growth of Clostridium
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        butyrate, thereby achieving aerobic growth of Clostridium tyrobutyricum.</p>
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    <p style="font-size: 180%; font-weight: bold;">Result</p>
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    <p>As can be seen from the sequencing results, the perR fragment has been knocked out.</p>
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    <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/h2/img1.png" width="80%" height="80%"></p>
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    <div align="center">
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        <strong>Fig2- Sequencing verification of obtained mutant. The red part represents the absence of the
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            sequence.</strong>
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    </div>
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    <p>At different rotational speeds, the growth of the plasmid knockout strain was better than that of the control
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        strain</p>
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    <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/h2/img2.png" width="80%" height="80%"></p>
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        <strong>Fig3- The growth profiles of Ct (ΔperR) and Ct (Control) under different contents of oxygen.</strong>
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    <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/h2/img3.png" width="80%" height="80%"></p>
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        <strong>Fig4- Physiological characteristics of Ct (ΔperR) and Ct (Control) in the presence of oxygen. (A) The
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            intracellular ROS levels in Ct (ΔperR) and Ct (Control) in the presence of oxygen. ROS levels were measured
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            in the logarithmic prophase. (B) The ratio of NADH/ NAD+ in Ct (ΔperR) and Ct (Control) in the presence of
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            oxygen. Phase I, II and III refer to the early-, mid- and late- logarithmic phase, respectively.</strong>
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    </div>
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    <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/h2/img4.png" width="80%" height="80%"></p>
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        <strong>Fig5- The survival rate of Ct (ΔperR) and Ct (Control) to oxidative stress.</strong>
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Revision as of 16:39, 11 October 2022


Plac-ΔperR

The upper and lower homology arms (HR-UP, HR-DOWN) were constructed at about 500 bp upstream and downstream of the PerR sequence of the Clostridium tyrobutyricum genome, plus a lactose-inducing promoter and a spacer sequence derived from the PerR , generating the CRISPR array .Then inserted it into the pMTL82151 plasmid, the successful plasmid is transformed into E. coli CA434, and introduced into Clostridium tyrobutyricum, by lactose induction to make the spacer hit the perR sequence on the target genome, thereby utilizing the endogenous CRISPR-Cas system knocks the perR out , eliminates the impediment effect of perR on the growth of Clostridium butyrate, thereby achieving aerobic growth of Clostridium tyrobutyricum.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 368
    Illegal PstI site found at 1597
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 368
    Illegal PstI site found at 1597
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 368
    Illegal BglII site found at 428
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 368
    Illegal PstI site found at 1597
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 368
    Illegal PstI site found at 1597
  • 1000
    COMPATIBLE WITH RFC[1000]


Document

Short Description: lactose-inducing promoter+ CRISPR array

The upper and lower homology arms (HR-UP, HR-DOWN) were constructed at about 500 bp upstream and downstream of the PerR sequence of the Clostridium tyrobutyricum genome, plus a lactose-inducing promoter and a spacer sequence derived from the PerR , generating the CRISPR array .Then inserted it into the pMTL82151 plasmid, the successful plasmid is transformed into E. coli CA434, and introduced into Clostridium tyrobutyricum, by lactose induction to make the spacer hit the perR sequence on the target genome, thereby utilizing the endogenous CRISPR-Cas system knocks the perR out , eliminates the impediment effect of perR on the growth of Clostridium butyrate, thereby achieving aerobic growth of Clostridium tyrobutyricum.

Result

As can be seen from the sequencing results, the perR fragment has been knocked out.

Fig2- Sequencing verification of obtained mutant. The red part represents the absence of the sequence.

At different rotational speeds, the growth of the plasmid knockout strain was better than that of the control strain

Fig3- The growth profiles of Ct (ΔperR) and Ct (Control) under different contents of oxygen.

Fig4- Physiological characteristics of Ct (ΔperR) and Ct (Control) in the presence of oxygen. (A) The intracellular ROS levels in Ct (ΔperR) and Ct (Control) in the presence of oxygen. ROS levels were measured in the logarithmic prophase. (B) The ratio of NADH/ NAD+ in Ct (ΔperR) and Ct (Control) in the presence of oxygen. Phase I, II and III refer to the early-, mid- and late- logarithmic phase, respectively.

Fig5- The survival rate of Ct (ΔperR) and Ct (Control) to oxidative stress.