Difference between revisions of "Part:BBa K3078110"

 
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<partinfo>BBa_K3078110 short</partinfo>
 
<partinfo>BBa_K3078110 short</partinfo>
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<h5>
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<P style="text-indent:2em;">
 
T4 holin regulated by tet-on system.When adding tetracycline or anhydroustetracycline, T4 holin expression turns on suicide system.  
 
T4 holin regulated by tet-on system.When adding tetracycline or anhydroustetracycline, T4 holin expression turns on suicide system.  
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</p>
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</h5>
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<h1>'''1. Usage and Biology'''</h1>
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<h5>
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<P style="text-indent:2em;">
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T4 holin is expressed after the suicide system is activated, which will lead to the non-specific damage of bacterial cytoplasmic membrane, forming stable transmembrane pores, and thus causing the death of bacteria.
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When there is no tetracycline, Ptet promoter will be inhibited by TetR, T4 holin will not be expressed and the bacteria survive. On the contrary, when tetracycline is added, TetR disinhibits Ptet and T4 holin is expressed, forming a stable transmeminal pore, leading to bacterial death.
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</p>
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</h5>
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<h1>'''2. Characterization'''</h1>
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<h4>'''2.1 Validation of pVE-TetR-T4 holin construction'''</h4>
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<h5>
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<P style="text-indent:2em;">
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To verify the construction of pVE-TetR-T4 holin which we generated, the digestion by SmalI was performed by a standard protocol followed by agarose gel electrophoresis (Figure 1).
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</p>
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</h5>
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[[File:自杀1.png|600px|center|自杀1]]
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<center>
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Figure 1. Digestion and electrophoresis of pVE-TetR-T4 holin.
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</center>
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<h4>'''2.2 Function of pVE-TetR-T4 holin '''</h4>
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<h5>
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<P style="text-indent:2em;">
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T4 holin is expressed after the suicide system is activated, which will lead to the non-specific damage of bacterial cytoplasmic membrane, forming stable transmembrane pores, and thus causing the death of bacteria.
 +
When there is no tetracycline, Ptet promoter will be inhibited by TetR, T4 holin will not be expressed and the bacteria survive. On the contrary, when tetracycline is added, TetR disinhibits Ptet and T4 holin is expressed, forming a stable transmeminal pore, leading to bacterial death. As the results indicated, after 24 hrs of 50 nM atc treatment, over 50% bacteria were lysed and dead, which attested that suicide system could work successfully (Figure 2).
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</p>
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</h5>
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[[File:自杀2.png|600px|center|自杀2]]
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<center style="text-align:left;">
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Figure 2. Verification of the suicide system. The plasmid was transformed into E. coli BL21, cultured overnight, and diluted to OD600 0.04. Anhydrotetracycline (atc) was added at OD600 0.9 to make the final concentration of atc reach 27 nM to measure at the indicated time. The experiment was performed three times in triplicate. *, P < 0.05 from control using Student’s t test. △, P < 0.05.
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</center>
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<h1>'''3. Conclusion'''</h1>
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<h5>
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<P style="text-indent:2em;">
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The function of this part has been successfully verified in our engineered bacteria, providing a practical suicide method.
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</p>
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</h5>
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Revision as of 22:51, 21 October 2019

T4 holin regulated by tet-on system.

T4 holin regulated by tet-on system.When adding tetracycline or anhydroustetracycline, T4 holin expression turns on suicide system.



1. Usage and Biology

T4 holin is expressed after the suicide system is activated, which will lead to the non-specific damage of bacterial cytoplasmic membrane, forming stable transmembrane pores, and thus causing the death of bacteria. When there is no tetracycline, Ptet promoter will be inhibited by TetR, T4 holin will not be expressed and the bacteria survive. On the contrary, when tetracycline is added, TetR disinhibits Ptet and T4 holin is expressed, forming a stable transmeminal pore, leading to bacterial death.


2. Characterization

2.1 Validation of pVE-TetR-T4 holin construction

To verify the construction of pVE-TetR-T4 holin which we generated, the digestion by SmalI was performed by a standard protocol followed by agarose gel electrophoresis (Figure 1).

自杀1

Figure 1. Digestion and electrophoresis of pVE-TetR-T4 holin.


2.2 Function of pVE-TetR-T4 holin

T4 holin is expressed after the suicide system is activated, which will lead to the non-specific damage of bacterial cytoplasmic membrane, forming stable transmembrane pores, and thus causing the death of bacteria. When there is no tetracycline, Ptet promoter will be inhibited by TetR, T4 holin will not be expressed and the bacteria survive. On the contrary, when tetracycline is added, TetR disinhibits Ptet and T4 holin is expressed, forming a stable transmeminal pore, leading to bacterial death. As the results indicated, after 24 hrs of 50 nM atc treatment, over 50% bacteria were lysed and dead, which attested that suicide system could work successfully (Figure 2).

自杀2

Figure 2. Verification of the suicide system. The plasmid was transformed into E. coli BL21, cultured overnight, and diluted to OD600 0.04. Anhydrotetracycline (atc) was added at OD600 0.9 to make the final concentration of atc reach 27 nM to measure at the indicated time. The experiment was performed three times in triplicate. *, P < 0.05 from control using Student’s t test. △, P < 0.05.



3. Conclusion

The function of this part has been successfully verified in our engineered bacteria, providing a practical suicide method.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]