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. It is induced by thermoinduction, as the presence of the colanic acid capsule means chemical or light triggers would not be as effective.

How

For the killing strategy, we needed to destroy the genetic material, whilst leaving the cell membrane intact - so as to maintain the protective capsule around our protein of interest.
Restriction enzymes are found bacteria and archaea, and act as defense mechanisms against invading viruses. They recognise a certain DNA sequence of a few bases, and cleave the DNA strand.The E.ncapsulator is engineered to manufacture restriction enzymes when triggered, and these will cleave the genetic material within into fragments - thereby killing the cell.
The restriction enzymes chosen were DpnII and TaqI, which both recognise a 4 base sequence of DNA for cleavage. They will therefore cut the genome at several different sites. As a protection mechanism against DNA destruction due to low levels of restriction enzyme production (before trigger), we have made use of the native E. coli dam methylase protection system. This methylates DNA, which means that only high levels of restriction enzyme (ie. upon trigger) will cleave the DNA.

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.