Module 3: Genome Deletion Overview

Restriction Enzymes

Restriction enzymes will cleave DNA at particular recognition sites. This leads to multiple double-stranded breakages in DNA, which is unlikely to be repaired in time and will subsequently result in cell death.

In our system, the restriction enzymes DpnII and TaqI are produced. DpnII and TaqI are 4 base cutters, specifically targetting and cutting the sequences GATC and TCGA respectively (refer to diagram).

4 base cutters were chosen as they have a higher frequency of cleavage. Assuming equal distribution of nucleotides, the probability of cleavage is (1/4)⁴, which means that on average the four cutter will on average cut every 256 base pairs. Given that the genome of E.coli is around 4 million base pairs, it will become totally digested.

With more cleavages, the repair system would not be able to cope with multiple cleavages, so the genetic material contained within the cell will all be destroyed, including any inserted DNA.

A distinct advantage of using restriction enzymes for our 'killing' mechanism is that the the genetic material is removed, but the cell membrane is left intact. Therefore, the protein of interest will still be protected by the encapsulated cell. This renders the bacterium no more than an inanimate shell containing our protein drug of interest.

Module 3 - Genome Deletion