Overview

What:

The aim of Module 3 is to neutralise any genetic material present within the cell and to ensure that the cells will not be able to cause harm to the consumer upon ingestion. The genetic material of the E.ncapsulator is cut up, leaving the cell membrane intact and the protein of interest contained within: in effect leaving a floating sack of protein contained within the secreted capsule.

Why

The E.ncapsulator requires that the E. coli should be dead upon ingestion. This will prevent any transfer of genetic material between the bacterium and any gut microflora present, thereby avoiding any unexpected pathogenic effects. This is also especially important if the E.ncapsulator is to attain public acceptance, due to concerns over genetically modified organisms.

When

The killing mechanism is only to be triggered once encapsulation is complete. This caveat brings with it certain restrictions in terms of trigger mechanisms that could be adopted in our system; the presence of the colanic acid capsule means chemical or light triggers would not be as efficient as when the capsule is lacking. To overcome this, genomic neutralisation is induced by thermoinduction.

How

The bacterial membrane is left intact so as not to disrupt the structure of the colanic acid layer. Any killing mechanism that completely destroys the bacterium would defeat the purpose of having a self-encapsulating drug production and delivery system. With this in mind, a strategy was devised that takes advantage of restriction enzymes DpnII and TaqI and the native E. coli dam methylase protection against these restriction enzymes. The dam methylation provides protection to the bacterium during drug production and encapsulation stages.

The λcI gene inhibits the λcI promoter under which DpnII and TaqI are controlled. Upon increasing temperature to 37°C - 40°C the λcI gene undergoes a conformational change and is no longer able to bind the λcI promoter. The promoter is then free to transcribe DpnII and TaqI and cleavage of the E. coli genome is induced, rendering the bacterium no more than an inanimate shell containing our protein drug of choice.