Part:BBa_K1197007

IPTG dependent B.subtilis Kill Switch construct

Kill switch mechanism:

For our kill switch system, we used a sense-antisense RNA mechanism under an IPTG inducible circuit. MazF which is a non-specific ribonuclease coding gene is used as killing agent against Bacillus subtilis. In our system, MazF is produced constitutively under Pveg promoter. Also the same Pveg promoter is responsible for constitutive LacI production. Anti MazF is an antisense RNA producing sequence which is reverse complement to first bases of MazF and its RBS sequence. Its production is regulated by pHyperspank promoter which is inhibited by LacI and reactivated by IPTG.

According to this kill switch mechanism, in a medium where there is no IPTG, LacI will bind to pHyperspank promoter and inhibit its work. By this way, Anti MazF antisense RNA cannot be produced. Therefore, MazF production will not be inhibited and it will kill bacteria. On the other hand, if there is IPTG in medium, it will bind to LacI and prevent inhibition of pHyperspank by LacI. As a result, Anti MazF system will work and an antisense RNA will be produced. Since this antisense RNA sequence will be reverse complement of mRNA produced from MazF gene, it will bind to this mRNA and prevent the protein expression. In this situation, though both Anti MazF and MazF mRNA’s will be produced, there will be no MazF protein expression and so bacteria will not die.

Characterization of kill switch mechanism:

To see if this kill switch system, and so Anti MazF, is working or not we performed transformation experiments with whole kill switch construct and spreaded on media which contain IPTG and without IPTG. Briefly, MazF construct (K1197006) and Anti MazF construct (K1197001) were assembled in pSB1C3 in E.coli. Then, this construct were amplified and inserted into B.subtilis vector, K823023. Then, this vector was linearized and transformed by electroporation. And it was plated on agar with or without IPTG. Also, overnight cultures of transformants in both LB liquid media w/IPTG and w/o IPTG was left overnight. And as theoretically expected, we saw that bacteria lived and reproduced in media which contain IPTG, while they cannot reproduce in media which do not contain IPTG in both liquid and solid media.

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]

B.subtilis transformation results of Kill Switch construct in solid and liquid media with or without IPTG: