Difference between revisions of "Part:BBa K3078110"
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[[File:自杀2.png|600px|center|自杀2]] | [[File:自杀2.png|600px|center|自杀2]] | ||
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| − | Figure 2. Verification of the suicide system. The plasmid was transformed into E. coli BL21, cultured overnight, and diluted to | + | Figure 2. Verification of the suicide system. The plasmid was transformed into <i>E. coli<i> BL21, cultured overnight, and diluted to OD<sub>600</sub> 0.04. Anhydrotetracycline (atc) was added at OD<sub>600</sub> 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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<h1>'''3. Conclusion'''</h1> | <h1>'''3. Conclusion'''</h1> | ||
Latest revision as of 23:45, 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).
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).
Figure 2. Verification of the suicide system. The plasmid was transformed into E. coli<i> 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. </center>
3. Conclusion
The function of this part has been successfully verified in our engineered bacteria, providing a practical suicide method.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]