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.

How was toximop made in the lab?

We were kindly gifted a pGEX6P plasmid containing PP1 from the Division of Signal Transduction Therapy at the University of Dundee.

To adhere to the iGEM rules and regulations, it was necessary to remove the specified illegal site present in PP1, Pst1. We introduced a missense mutation through site directed mutagenesis of adenine 900 base to be substituted by cytosine without disturbing the alanine amino acid towards the N terminus of the protein. This was done by designing appropriate primers and a method of polymerase chain reaction (PCR). The PCR product was digested using restriction endonuclease Dpn1 as it recognises methylated sites, therefore recognising the original sequence and destroying it, leaving only mutated PP1 fragments. It was then cloned into both pUniprom and pSB1C3 plasmids and grown in E.coli DH5α cells.

In order to carry future tests on PP1, we decided to fuse it to a HA-tag (a DNA sequence derived from human influenza hemagglutinin) which can be detected by antibodies. This was carried out at the C terminus of PP1 by the use of PCR, digestion with XbaI/HindIII (enzyme) and ligation.

TorA is a TMAO reductase. Its signalling sequence is often used in research, as it is a twin-arginine translocation (Tat) pathway signal and it relies on pre-translocation protein folding in the cytoplasm. TorAss was obtained from the Division of Molecular Microbiology.

MalE (Maltose binding periplasmic protein) is involved in the high-affinity membrane transport system, MalEFGK, which uses the Secretory transport pathway (Sec). In contrast to Tat, Sec relies on post-translocation protein folding and in some cases it also pushes protein into the inner membrane of bacteria. malEss was obtained by PCR from the chromosomal DNA of E.coli MG1655.

PrsA (penicillin binding protein) is membrane bound and it is present at distinct spots on the membrane forming a helical pattern. Its main role is to catalyse post-translocation folding of membrane proteins and it is also essential for normal growth of B. subtilis ; prsAss was amplified by PCR using appropriate primers from B. Subtillis strain 3610.

All of the signalling sequences were cloned into pUniprom by themselves and after sequencing they were fused at the N-terminus of HA-tagged PP1. This is only useful for the E.coli ToxiMop, as pUniprom is a plasmid unsuitable for use in B. subtilis. PrsAss-PP1-HA had to be cloned into pDR110 vector, which would then recombine into the AmyE locus of the chromosomal DNA creating the B. subtilis ToxiMop.

To test the efficiency of the ToxiMop we had to test: expression and cell targeting, binding of microcystin, dialysis bag

In E. coli, expression of PP1 was tested by cell fractionation of the bacterial strain expressing PP1 fused to a HA tag for Western Blot analysis.

• Whole Cell Control (sample of overnight)
• Supernatant (to test if PP1 is being secreted)
• Whole Cell
• Periplasm
• Sphaeroplast
• Cytoplasm
• Membrane

These fractions were then used in Western blots to test where in E. coli PP1 is being targeted to.

To determine the best targeting mechanism in E. coli, export of PP1 to the periplasm was tested with MalE (Sec) or TorA (Tat) signal sequence (Figure ). Describe results from western blot. Since PP1 transport is more efficient via the Tat pathway, the construct pUniprom-TorA(ss) PP1 HA has been used for all further characterization experiments of PP1.

Later blots of only MalE fractions suggested that PP1 was not being exported to the periplasm, but may be getting stuck in the membrane during Sec transport (figure) .

Further characterisation of export via the Tat pathway was carried out by fractionation and blotting of normal wild type cells expressing TorA:PP1:HA versus mutant E. coli cells that do not have a Tat pathway (∆Tat) expressing TorA:PP1:HA.