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Imperial College London/M3/Detail
From 2009.igem.org
Module 3
Killing Strategy
Our design for The E.ncapsulator requires the cell to be dead upon ingestion. This was decided for a number of reasons. Firstly, destroying the genetic 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 consumption of genetically modified organisms.
It is also necessary to make sure the cell membrane is not disrupted, so our polypeptide of interest remains protected. Any disruption to the membrane would result in the polypeptide being susceptible when exposed to the acidic conditions of the stomach. Restriction enzymes seemed to be a good way of ensuring that the cell is no longer viable, whilst leaving the cell membrane intact.
Restriction enzymes are enzymes that recognise and cut specific DNA sequences. They are naturally produced by E.Coli as a defense against invading viruses. Different restriction enzymes can cut different sequences of varying sizes. The E.ncapsulator uses two restriction enzymesdpnII and taqI to cleave the genetic material within into fragments - thereby killing the cell.
As a protective mechanism against DNA destruction due to basal levels of restriction enzyme production, 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.
is induced, rendering the bacterium no more than an inanimate shell containing our protein drug of choice.
Induction
Once encapsulation is completed, there must be some way of inducing the production of restriction enzymes. Any chemical inducers may be ineffective if they cannot diffuse through the colanic acid capsule. We therefore decided on using a temperature inducible system. This means that Module 3 can be triggered once the encapsulation is sufficiently complete, only the temperature needs to be raised. This allows complete control by the user as to when Module 3 begins.
This temperature sensitive mechanism makes use of the p-lamda promoter. Once the temperature is raised above 42 degrees Celsius the promoter is induced, resulting in expression of our restriction enzymes.
