Part:BBa_K3078110

T4 holin regulated by tet-on system.

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

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]