Module 2: Aggregation

Description | Design | Construction | Characterisation | Shear Device | Modelling | Results | Conclusions

Description

The Aggregation Module is a means of conferring onto our bacteria the ability to construct islands from smaller plastic fragments. To do so we have decided to transform our bacterium with a circuit to produce an adhesive protein called Curli. As Curlis are non-specific in the surfaces they bind, we also have a module (Module 1) for limiting their production unless they are in the presence of plastic.

Production of Curli is dependent on activation of the Sal Operon promoter of Module 1. In our system Curlis will encourage adhesion of bacteria to the plastic fragment, and assist the formation of biofilms. Adhesion between biofilm-covered plastic fragments will allow smaller plastic fragments to aggregate into larger plastic formations.

Curli formation involves a large number of genes under the control of an operon promoter. The BioBrick we will be using (BBa_K540000) carries a Cobalt promoter, which in our system will be switched for the p(Sal) Operon (BBa_ described in Module 1.

How are Curli produced? Curli production is complex, and not fully explained. It is known that there are two key structural proteins, CsgA and CsgB, which are reasonably well characterised. CsgA is the main structural component, which is a secreted from the cell as subunits. CsgB is also secreted, and is essential to allow the CsgA subunits polymerise (assemble) into fibrils. This is under the control of the CsgAB operon.

A second operon CsgDEFG, containing four regulatory genes, is also required for Curli production. The first of these a transcriptional regulator called CsgD drives transcription of the genes of the CsgAB operon. Less is known of CsgE and CsgF of the CsgDEFG operon, but they appear to regulate or stabilise somehow the fourth member of the operon – CsgG. CsgG is an outer membrane lipoprotein, which interacts with itself to form an oligomeric ring-shaped structure through the membrane. This acts as a pore to allow CsgA and CsgB to leave the cell, to form fibrils.

All of these constituents will have to be transformed into our bacteria, but where bacteria naturally regulate Curli production to respond to their environment, our system will place the production of Curli under the control of the Detection Circuit (Module 1).