Introduction

We aim to provide a novel chassis with improved bio-safety capabilities. This will take the form of a Maxicell - achromosomal E. coli - which is incapable of reproducing or of performing horizontal gene transfer. In order to prevent horizontal gene transfer we intend to take a two pronged approach; Semantic Containment will mean that other organisms should not be able to 'read' the DNA of the Maxicell if they should take it up and a DNA degrading killswitch will break up the Maxicell plasmid/s after a given time span.

DNA Degradation Switch Concept

The implementation of our DNA degradation system requires 2 constructs:

• Colicin E2 plasmid (pCol) - Colicin E2 is a nicking endonuclease which acts without specificity to cut both single and double stranded DNA. This plasmid will contain Constitutive Anderson Promoter, RBS, Colicin E2 coding sequence and T1 Terminator.
• Immunity Plasmid - Imm2 is the immunity protein for Colicin E2. Imm2 binds to Colicin E2 with high affinity and when present in equimolar concentration prevents its DNase activity. This plasmid will contain Constitutive Anderson Promoter, RBS, Imm2 Coding Sequence and T1 Terminator. In addition a site for the homing endonuclease I-SceI will be present that when cut turns off Imm2 expression.

Prior to Maxicell formation our E. coli will be transformed with pImm, this will cause a build up of Imm2 within the cell. At this phase of the model only equations for Imm2 synthesis are evaluated.

Once Imm2 concentration has reached equilibrium the E. coli can then be transformed with pCol. Colicin E2 will be expressed within the cell however the build-up of Imm2 and its continued expression ensure it remains inactive. At this phase in the model all equations are evaluated.

In order to break up the chromosome of our E. coli and form Maxicells we have obtained a strain from Leach Lab containing a number of I-SceI sites within the chromosome. When I-SceI is introduced and Maxicells are formed pImm will also be cut 'turning off' expression of Imm2. At this phase in the model all equations are evaluated however rate of imm transcription is 0 and halflives for Imm2 and Colicin E2 are altered to reflect that replication is no longer taking place.

By modeling this simple killswitch we can determine the plausibility of the system as well as screen across different Anderson Promoters and RBS to find the killswitch activation time closest to the length of time for which Maxicells can continue expressing protein.

Ordinary Differential Equations

The model uses a simple set of ordinary differential equations:

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