Team:Dundee/Project/Mop

The ToxiMop are two bacterial strains that have been designed to clean microcystin from contaminated water. These strains have been engineered to express human PP1, to which microcystin binds covalently, and target it outside the bacterial membrane to allow easy binding of the toxin. This binding inactivates the toxic activity of microcystin, effectively ‘mopping’ it up.

There are currently two chassis in which PP1 is being expressed: Bacillus subtilisand Escherichia coli. These two organisms were selected as they have different membrane layouts and allowed us to take two different approaches to the mop. Localisation of PP1 is mediated via different signal sequences that direct PP1 to the periplasm in E. coli and membrane in B. subtilis.

We considered two protein transport pathways for the transport of PP1 in our mop, the Sec and Tat protein transport pathways. We took these into consideration as Sec deals with unfolded protein (inserts membrane proteins into the inner membrane) and Tat deals with folded protein. In this way we could find out if PP1 was folding correctly in the periplasm by comparing blots of samples using Sec and samples using Tat.

As B. subtilis is Gram positive, PP1 is transported to the membrane. In this instance, export of PP1 is mediated by the Sec protein transport system, towards this aim; we fused PP1 to the PrsA signal sequence.

E. coli is Gram negative and PP1 is targeted to the periplasm. Transport of PP1 to the periplasm can be mediated by either the Sec or Tat protein transport systems. For our project, we chose the MalE (Sec) and TorA (Tat) signal sequences.