Experiments and
Plasmid Design

Darobactin

Darobactin targets the bamA complex of gram-negative bacteria. bamA is a component of the β-barrel assembly machinery (bam) complex for proper folding and insertion of outer membrane proteins. Darobactin selectively binds to the lateral gate of bamA, disrupting formation of the bacterial outer membrane, leading to cell death. Darobactin stabilizes a closed lateral gate conformation upon binding to bamA, preventing proteins from integrating into the outer membrane. Darobactin functionality depends on the formation of a macrocyclic ring.

Darobactin is a ribosomally synthesized and post-translationally modified peptide. The dar gene cluster encodes darA, a 58 amino acid precursor peptide, and four proteins, a radical S-adenosylmethionine (rSAM) enzyme darE and three ABC transporter proteins darBCD. darE constructs the bicyclic ring system of darobactin by installing ether and C-C crosslinks. Then, the leader and tail regions of darA are cleaved off by a peptidase and darobactin is secreted by darBCD.

Our Composite Plasmid

Using BBa_K5145005 (a mutated bamA sequence) and a repressible promoter as a kill switch system

BamA encodes the part of the BAM complex that is responsible for the synthesis of 𝛽-barrel proteins in the bacteria’s outer membrane. Darobactin is able to target BamA and compromise the integrity of the bacteria’s outer membrane, which would lead to cell death.
The expression of darABCDE can be regulated through an environmentally sensitive promoter to ensure the darobactin is produced only under desired conditions. By using this promoter for expression of both darABCDE and bamA, their expression can be coordinated. (See promoter page)
This is because while darobactin is being produced by the cell, expression of bamA allows the cell to be resistant to the compound. However, when the promoter is turned off, the cell will still be encompassed by darobactin, while bamA is no longer expressed, leading to cell death. This process acts as a kill switch. To ensure that bamA is expressed, we chose to use a mutant bamA rather than the wildtype. This involved engineering our plasmid to include the mutant bamA that will be inserted into the bacterium. The mutant bamA should strongly express itself, overpowering the wildtype bamA and resulting in a non-functioning bamA, in theory, though this theory will need to undergo extensive testing for confirmation.