Module 2 - Encapsulsation

Overview

Module 2: Encapsulation

Module 2 Initiation:

Module 2 is triggered once levels of intracellular cyclic AMP (cAMP) rise. This is becuase cAMP facilitates the dimerisation of the transcription factor CAP which induces transcription from the PcstA promoter. The rise in cAMP is correlated with a fall in levels of the primary carbon source (glucose). Thus, by controlling the initial amount of glucose present in the system, we achieve a timer function for the start of Module 2.

It should be noted that Module 1 polypeptide production continues at a lower 'maintenance level' throughout Module 2.

Module 2 Acid Resistance:

Encapsulation (Module 2) allows the safe passage of polypetides through the stomach into the intestine. Without encapsulation, our polypeptides would be denatured and degraded by stomach acid and digestive proteases respectively.

By 'hacking' E.coli’s endogenous acid resistance pathways in three places we induced the formation of a safe colanic acid microcapsule that protects our chassis from the harsh acidic conditions of the stomach.

The Encapsulation Triple Hack:

1. RcsB up regulation = Induces colanic acid biosynthesis as well as many other membrane fortifying genes.

2. YgiV up regulation = Blocks biofilm formation and enhanced colanic acid production.

3. Rfal up regulation = Tethers colanic acid to the cell surface creating a microcapsule.

Once The E.ncapsulator reaches the intestine, gut microflora will digest the colanic acid coat, resulting in the release of the polypeptide therapeutic. Colanic acid represents a rich sugar source and many gut bacteria such as Citrobacter have been found to contain the necessary enzymes to break it down. In addition, a number of phages have a similar enzyme attached to their tail fibers which allows them to break into colanic acid coated cells.

Module 2 Freeze Drying:

In addition, Module 2 involves the synthesis of the preservative trehalose. This is a disaccharide that allows us to freeze dry the chassis without damaging either its structural integrity or the intracellular polypeptides. OtsA and OtsB are the two genes required for trehalose production. We hope that by introducing additional copies of these genes on a plasmid we can boost trehalose production. Freeze drying is an important storage mechanism that will facilitate the widespread dispersal of E.ncapsulator technology.

Module 2 End:

The end of Module 2 occurs when the temperature of the culture is raised to 42 degrees centigrade. This temperature shift triggers Module 3 in which the genome is deleted.

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Module 2 Contents