Part:BBa_K2757001

Art-175 fused with N-terminal DsbA 2-19 signal peptide and bidirectional pTet

This part consists of the artilysin Art-175 with a DsbA signal sequence under the influence of a bidirectional pTet promoter. Intracellular production of antimicrobial Art-175 commences upon induction of the tetracycline-regulated promoter. The DsbA secretion tag facilitates cotranslational export of Art-175 into the periplasmic space via the SRP pathway, promoting subsequent lysis of the host cell. The part was designed as a kill switch to promote host cell lysis upon interaction with a synthetic TetR inducer.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 86
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 534
  Illegal AgeI site found at 733
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

The graph on the left displays Art-175-DsbA with bidirectional pTet promoter. The graph on the right depicts the Art-175-DsbA construct without a promoter (BBa_K1659002); no significant difference in growth is seen relative to the control vector without Art-175 in the presence of ATC.

Bidirectional pTeT Controlled Kill Switch Growth Assay

The kill switch was assayed by measuring the OD of cultures subjected to varying levels of the inducer. While not intended to be used in final probiotic, the tetracycline analogue, anhydrotetracycline (ATC), has been shown to have a lower antibiotic activity and affinity for TetR and was thus chosen for use in our experiments. By comparing the growth of cells transformed with a plasmid lacking the promoter and cells with a plasmid lacking the killswitch and the promoter, we showed that cell growth was not affected by the kill switch.

Cell growth was unaffected by leaking of the promoter in cells with the bidirectional kill switch construct, showing that our device will not restrict the ability for the chassis to colonise the target region of the gut.

Our plate reader assay shows that the bidirectional promoter/kill switch construct induces cell lysis in response to concentrations of ATC equal to or above 1nM. Higher concentrations increase the rate of cell lysis up to concentrations of 20nM. Above this concentration, the promoter is at maximum activity.

Cell growth for negative controls, while higher than cells with the kill switch construct, was reduced by high ATC concentrations. This shows that while ATC has less antibiotic activity than tetracycline, an analogue with lower antibiotic activity or increased binding affinity for TetR is required for a highly selective and effective inducible kill switch.